The high-mobility group A (HMGA) nonhistone chromosomal proteins HMGA1 and HMGA2 play a role in determining chromatin structure and in regulating the transcription of several genes. High levels of these proteins are characteristic of rapidly dividing cells in embryonic tissue and in tumors. The aim of this study was to determine the role of HMGA1 and HMGA2 throughout mouse spermatogenesis. Northern blot analysis and immunocytochemistry showed HMGA1 and HMGA2 expression during the progression from spermatocyte to spermatid. Interestingly, Western blot analysis with antibodies against the HMGA1 gene product revealed only the HMG1c isoform (27 kDa) in the testis; HMGA1a and HMGA1b were undetectable. These three isoforms are encoded by the HMGA1 gene through alternative splicing. Finally, few spermatids and complete absence of spermatozoa were observed in the testes of HMGA2-null mice, which suggests that the HMGA2 gene plays a critical role in male fertility.