OBJECTIVE Factors governing events between exposure of male genital mucosa surfaces and the establishment of infection are poorly understood. Furthermore, little is known about the safety and efficacy of microbicides on male genital mucosa. DESIGN Here we present a novel penile tissue explant model to characterize the mechanisms of HIV-1 infection of male genital tissue and evaluate candidate microbicides. METHODS Mucosal explant culture conditions were determined for glans, urethra and foreskin obtained from gender reassignment and circumcision. Density and distribution of CD4 and CD1a cells were visualized by microscopy. In vitro HIV-1 infection was determined by measuring p24 release, whereas microbicide biocompatibility and efficacy were assessed by measurement of tissue viability, cytokine expression and p24 production. RESULTS Cultured glans and foreskin showed comparable epithelial thickness but some differences in CD4 and CD1a cell density. All tissue sites examined (foreskin, glans, meatus, urethra) were equally susceptible to R5 HIV-1 infection, which was productively disseminated by migratory cells emigrating from tissue. In contrast, X4 HIV-1 failed to infect mucosal tissue and dissemination by migratory cells was less efficient. The three candidate microbicides poly(methyl 2-propionamidoacrylate), PRO 2000 and Cyanovirin-N, showed good tissue compatibility and efficient prevention of HIV-1 infection, causing only minor changes in tissue cytokine profile. CONCLUSION The described model provides a useful model to study the determinants of HIV-1 infection of male genital tissue and is likely to be an important tool for the future development of microbicide candidates and concepts.