The gonad-specific expression of a recently discovered Igf subtype (Igf3) in teleost has indicated the important role of this novel Igf in the reproductive functions of fish. In the present study using zebrafish as the model organism, we have further examined the gene expression patterns and the physiological role of Igf3 in the ovary. The igf3 gene in zebrafish was found to be alternatively spliced into two transcripts, with transcript variant 1 exclusively expressed in the gonads, and transcript variant 2 only expressed during early development. Using specific antibodies developed for zebrafish Igf3, both the Igf3 prepropeptide and the mature peptide forms of Igf3 were found to be predominantly expressed in the zebrafish ovary. Real-time PCR and in situ hybridization revealed that igf3 mRNA is relatively low in the early follicles, but is significantly increased after the mid-vitellogenic stage (midstage III), and is high in the full-grown follicles. In the full-grown follicles, igf3 mRNA was detected mainly in the somatic follicular cells with a low level of expression in the oocytes. Igf3 immunoreactivity was confined to the follicular cells only. The expression of igf3 was significantly up-regulated in both ovarian fragments and isolated follicles upon treatment with human chorionic gonadotropin in dose- and time-dependent manners. Treatment with 8-bromoadenosine 3′,5′-cyclic monophosphate or 3-isobutyl-1-methylxanthine also up-regulated the expression of igf3 in full-grown follicles. Incubation of follicles with recombinant zebrafish Igf3 significantly enhanced oocyte maturation in time-, dose-, and stage-dependent manners. The actions of Igf3 could be blocked by cycloheximide, but not by actinomycin D. Taken together, these results support an important role of Igf3 in the ovarian functions of zebrafish, especially in oocyte maturation.