The current study examines the expression and potential actions of neurotropin-3 (NT3), nerve growth factor (NGF), and their receptors during morphological sex determination (seminiferous cord formation) and perinatal rat testis development. The expression of neurotropins and their receptors was analyzed with immunohistochemistry. Cellular localization of neurotropin ligand and receptor proteins changed during embryonic testis development. Neurotropin-3 was localized to Sertoli cells at Embryonic Day 14 (E14), was present in gonocytes at Postnatal Day 0 (P0), and after birth became localized to the interstitium and Sertoli cells (P3–P5). The expression of trk C (the high affinity receptor for NT3) was localized to mesonephric ducts and cells surrounding the cords (E14–E18). In addition, Sertoli cells and preperitubular cells surrounding the cords at E14 also stained for trk C. Neurotropin-3 was expressed in gonocytes and Sertoli cells at P0–P5. Nerve growth factor was detected in Sertoli cells at E14, was clearly in Sertoli and interstitial cells at E16 and E18, and in Sertoli, germ, and interstitial cells from P0–P5. The expression of trk A (the high affinity receptor for NGF) was located in Sertoli and interstitial cells at E16–P5. To determine the actions of neurotropins during embryonic and perinatal testis development, experiments were conducted on E13 and P0 testis. Antisense oligonucleotide experiments with NT3 were used on E13 testis organ cultures to determine effects on seminiferous cord formation. Cord formation was inhibited in 40% of the organ cultures treated with the antisense NT3 oligonucleotides, while no inhibition was observed with sense oligonucleotides. In P0 testis cultures, both NT3 and NGF alone and in combination stimulated thymidine incorporation into DNA. Therefore, the neurotropins are involved in embryonic morphological events (cord formation; NT3) and in growth of the perinatal testis (P0; NT3 and NGF). To define further the growth effects of neurotropins on testis development, expression of transforming growth factor alpha and beta (TGFα and TGFβ) were examined in response to neurotropins. The P0 testis cultures were treated with neurotropins, and expression of mRNA for TGFα and TGFβ was analyzed utilizing a quantitative reverse transcription-polymerase chain reaction assay. Nerve growth factor and NT3 alone or in combination inhibited expression of mRNA for TGFα while NT3 increased mRNA expression of epidermal growth factor receptor. The combination treatment of neurotropins inhibited expression of TGFβ1 and increased expression of TGFβ3. In summary, observations suggest that NT3, NGF, trk A, and trk C are localized to cells critical to seminiferous cord formation and appear to be important regulators of morphological sex determination. In addition to these morphological effects, both NT3 and NGF stimulate P0 testis growth and may elicit their action through altering the expression of locally produced growth factors such as TGFα and TGFβ. Taken together these results suggest that neurotropins are regulators of paracrine cell-cell interactions that result in morphological sex determination and perinatal testis growth.