A method for culturing endothelial cells (HCC-EC) from surgical specimens of human corpus cavernosum has been developed. The approach involves selective endothelial outgrowth from explants and may be generally applicable to tissues whose endothelium is not amenable to isolation by routine mechanical or enzymatic methods. The tissue is minced into pieces which are placed onto gelatin- or fibronectin-coated tissue culture plastic, and grown in medium suitable for microvascular endothelial cell growth (Carson and Haudenschild, In Vitro 22:344-354, 1986). By Days 5 to 7 EC colonies are found. Within a day or two after the appearance of the EC colonies, a non-EC cell type appears and, if undisturbed, quickly overgrows the EC. An exploitable temporal separation between the emergence of EC and non-EC is obtained when both conditioned medium (from bovine aortic endothelium) and retinal extract are present during the outgrowth period. Explants are removed by pipetting at the first sign of the emergence of the non-EC cell type. Once isolated, HCC-EC do not require conditioned medium but do require either retinal extract or acidic fibroblast growth factor for survival and growth. Approximately 60% of the first passage cultures are at least 80% EC as judged by DiI-Ac-LDL labeling. One corpus (0.3 x 0.3 x 0.5 cm) usually produces 120 cm2 of primary culture within 2 wk. These EC form contact-inhibited monolayers and stain positively for Factor VIII. They have a doubling time at 6th passage of 48 h and a plateau density of 5 to 7 x 10(4) cells/cm2. The availability of such cultures should facilitate the study of endothelium-mediated responses which play an important role in the erectile function of human penile corpus cavernosum.