Germ cell nuclear factor (GCNF/RTR), a novel orphan receptor in the nuclear receptor superfamily of ligand-activated transcription factors, is expressed predominantly in developing germ cells. In several mammalian species two GCNF/RTR mRNAs are present in the testis, with the smaller 2.3-kb transcript generally expressed at higher levels than the larger 7.4- or 8.0-kb transcript. In both the mouse and rat, the 2.3- and 7.4-kb GCNF/RTR transcripts were detected in isolated spermatogenic cells, but not in Sertoli cells. Expression of these transcripts is differentially regulated, with the larger 7.4-kb mRNA appearing earlier during testicular development. The major 2.3-kb transcript is expressed predominantly in round spermatids in the mouse and rat. In situ hybridization studies in the rat demonstrated that GCNF/RTR transcripts reach maximal steady-state levels in round spermatids at stages VII and VIII of the spermatogenic cycle, and then decline abruptly as spermatids begin to elongate. RNase protection assays were used to predict the 3' termination site of the 2.3-kb transcript. An alternative polyadenylation signal (AGUAAA) was identified just upstream of this termination site. These studies suggest that GCNF/RTR may regulate transcription during spermatogenesis, particularly in round spermatids just prior to the initiation of nuclear elongation and condensation.