It has recently been determined that the intracellular calcium receptor, calmodulin, is encoded by a multigene family. At least three calmodulin genes that encode the identical protein are expressed in adult testis from a variety of mammalian species. The complementary techniques of RNA blot hybridization and in situ hybridization were used to assess the relative levels of calmodulin RNAs during rat testis cell differentiation. RNA isolated from highly purified populations of somatic or germinal cells and sections of fixed testis were analyzed for hybridization to specific probes corresponding to the three separate rat calmodulin genes. The level of each calmodulin RNA can be described by a unique developmental pattern during spermatogenesis. The steady state levels of transcripts corresponding to calmodulin gene I (CaM I) and III increase in early meiotic cells (leptotene-zygotene spermatocytes) and remain constant throughout meiosis. The level of the CaM II RNA increases between early and mid-pachytene spermatocytes, but is only transiently elevated. The level of this RNA decreases in round spermatids and is almost nondetectable in cytoplasts shed from elongating spermatids. The levels of CaM I RNAs are maintained in early round spermatids, but only RNA derived from the CaM III gene is still evident in late spermatids. The data indicate that an individual germ cell contains RNAs derived from multiple calmodulin genes and suggests that the divergent calmodulin genes may respond to different cellular regulatory signals.