The neural cell adhesion molecule (NCAM) polypeptides are expressed by numerous tissues during embryonic development, where they are involved in cell-cell interactions. In the adult, NCAM expression is confined to a few cell types, including neurons and peptide-hormone-producing cells. Here we demonstrate that the Leydig cells of the adult rat, mouse, and hamster testes express NCAM as well. Western blotting showed that an NCAM of approximately 120 kDa was present in the adult testes of all three species investigated. This form was also found in freshly isolated mouse Leydig cells and in Leydig cells after 2 days in culture. After 4 days in culture, mouse Leydig cells expressed additional NCAM isoforms of approximately 140 and 180 kDa, indicating changes in alternative splicing of NCAM primary transcripts. Also, NCAM mRNA of all isoforms, as detected by S1-nuclease protection assays, increased with time in culture. The expression of the cell adhesion molecule NCAM by adult Leydig cells may explain the aggregation of Leydig cells in clusters in rodent testes, which could be a prerequisite for functional coordination of groups of Leydig cells. Furthermore, the presence of this neural and endocrine marker may indicate a closer relationship between Leydig cells and neural and peptide-hormone-producing cells than is considered to exist at the present time.