Losses of human melanocytes (HM) in transplantation of cultured skin substitutes (CSS) may result from poor cellular attachments. To test this hypothesis, HM integrin expression was measured in four culture media: (a) melanocyte growth medium (MGM), an HM proliferation medium; (b) UCMC 160, a CSS maturation medium; (c) mMGM, modified MGM with 1.8 mM calcium; and (d) modified UCMC 160 with HM supplements (mUCMC 160). HM grew well in all media except UCMC 160. Increased expression of β1, β4, α3β1 and α5 integrins on HM cultured in MGM and mMGM versus UCMC 160 was found by flow cytometry. Annexin V-allophycocyanin (APC) labeled HM in apoptosis and increased significantly in UCMC 160 (31.1%) compared with MGM (11.9%) or mMGM (13.9%). CSS were incubated in UCMC 160, mMGM or mUCMC 160 media, and grafted to athymic mice. In the mMGM group, grafts were darker as measured with a chromameter through 6 weeks and the average number of basal HM per field was greater at 12 weeks post-grafting. Increased graft loss was observed in the mMGM group which corresponded with the poor epidermal morphology in vitro. Although HM retention improved in vivo using mMGM to culture the CSS, the stability of the epidermis decreased. These results indicate that expression of integrins on HM in vitro correlates with HM retention in CSS and short-term survival after transplantation, but that long-term survival depends also on stable epithelium.