Recently, it was found by two research groups that LY6A, known widely in the stem cell community as stem cell antigen-1 or SCA-1, is expressed on testicular side population (SP) cells. Whether these SP cells are spermatogonial stem cells is a point of disagreement and, therefore, the identity of the LY6A-positive cells as well. We studied the expression pattern of LY6A in testis by immunohistochemistry and found it to be expressed in the interstitial tissue on peritubular myoid, endothelial, and spherical-shaped peritubular mesenchymal cells. To address the question whether LY6A has a function in spermatogenesis or testis development, we studied the testis of Ly6a−/− mice (allele Ly6atm1Pmf). We found no morphological abnormalities or differences in numbers of spermatogonia, spermatocytes, Leydig cells, or macrophages in relation to the number of Sertoli cells. Therefore, we conclude that LY6A expression does not influence testis development or spermatogenesis and that spermatogonial stem cells are LY6A negative.