AbstractTargeted mutagenesis of the retinoic acid receptor α (RARα) gene has revealed its essential role in spermatogenesis. Although cells in all stages of spermatogenesis were detected in RARα‐/‐ testes, there was an increase in degenerating pachytene spermatocytes and a temporary developmental arrest in step 8–9 spermatids in the first wave of spermatogenesis, a delay in the onset of the second wave, and a temporary arrest in preleptotene to leptotene spermatocytes in the first, second, and third waves. A striking aspect of the mutant phenotype was the failure of spermatids to align at the tubular lumen at stage VIII. Furthermore, there were missing or decreased numbers of the predicted cell types in tubules, and they exhibited a profound asynchrony of mixed spermatogenic cell types. In vivo bromodeoxyuridine labeling revealed a significant decrease in germ cell proliferation in both juvenile and adult RARα‐/‐ testes and confirmed the arrest at step 8–9 spermatids. Retinoid signaling through RARα, thus, appears to be critical for establishment of synchronous progression of spermatogenesis and the subsequent establishment of correct cellular associations. Developmental Dynamics 230:754–766, 2004. © 2004 Wiley‐Liss, Inc.