AbstractHedgehog (Hh) signalling is known to regulate many aspects of normal development as well as being upregulated in various cancers. Signalling is mediated by the Gli family of zinc finger transcription factors. Based on observations that deletion of one of the three Hh genes,Dhh, leads to male infertility, we hypothesized that regulated expression of Hh signalling components would be a feature of adult spermatogenesis. We used in situ hybridization to characteriseGligene expression in juvenile and adult mouse testes. In the first wave of spermatogenesis, mRNAs encoding all threeGlisare detected in spermatogonia and Sertoli cells. In adult mouse testes, these transcripts are observed in spermatogonia and spermatocytes, with reduced signal intensity in round spermatids. The mRNAs encoding key effectors of Hh signalling,Ptc2, Smo, andFu, are also most apparent in spermatogonia, spermatocytes, and to a lower extent in round spermatids. In contrast, mRNA encodingSuFu, a negative regulator of Hh signalling, was most predominant in round spermatids and the protein is evident in round and elongating spermatids, suggesting that SuFu protein may switch off Hh signalling in haploid germ cells. Overall, the coordinated expression pattern of these genes in adult mouse testis indicates a role for Hh signalling in spermatogenesis.Developmental Dynamics 235:3063–3070, 2006. © 2006 Wiley‐Liss, Inc.