In order to better understand the implications of gap junction proteins in spermatogenesis, connexin 43 (Cx43), the most abundant connexin in the testis, was evaluated in testes of wild-type mice and of two mutants with impaired spermatogenesis (ebo/ebo, and jun-d-/-mice). Reverse transcription-polymerase chain reaction (RT-PCR) amplification revealed a constitutive expression of mRNA for Cx43 in both wild-type mice and infertile mutants. In the seminiferous tubules of wild-type mice, indirect immunofluorescence revealed that Cx43 expression was stage-dependent and that the signal was mainly located in the region of Sertoli cell occluding junctions. Colocalization of Cx43 and of the tight-junction-associated protein zonula occludens 1 (ZO-1) was demonstrated in seminiferous tubules by using dual-label immunofluorescence in conjunction with confocal microscopy. The Cx43 staining analyzed by high-resolution confocal microscopy appeared as continuous, anastomozed ribbons and thin dots. The level of Cx43 immunoreactivity was reduced in seminiferous tubules of ebo/ebo and jun-d-/- mutants as compared to the respective wild-type mice. No staining for Cx43 was detected in Sertoli cell-only seminiferous tubules observed sometimes in jun-d-/- mice. The present study represents one of the first in vivo examples of alteration of seminiferous tubule Cx43 in testes with impaired spermatogenesis.