It was recently discovered that murine epidermal Langerhans cells (LC) changed significantly in function and phenotype when maintained in culture. Notably, accessory cell function for primary immune responses increased while cytologic markers like ATPase, nonspecific esterase, and Birbeck granules were lost. To further analyze LC differentiation, we used flow cytometry and a panel of 22 monoclonal antibodies to quantitate changes in surface antigens at the single-cell level. A striking change was a fivefold increase in the amount of Ia antigens (which are expressed on class II MHC products) during the first day of culture. The increase was evident within 3 h and reached a plateau at 15-24 h. Both I-A and I-E products behaved similarly. The increase in Ia was blocked by 1 microgram/ml cycloheximide. Expression of other surface antigens was then monitored on Ia+ LC by two-color flow cytometry. Low levels of class I (H-2D and H-2K) MHC products were detected on freshly isolated LC, and these antigens also increased severalfold during the first day of culture. Fc receptors (identified with the 2.4G2 mAb) and the F4/80 macrophage antigen decreased, as reported previously. Three antigens that were detected in fresh suspensions were expressed at constant levels in culture. These were the C3bi receptor and the pan leukocyte and interdigitating cell antigens. Several leukocyte antigens that were not found initially on LCs did not appear, including B220 anti-B cell, 33D1 anti-dendritic cell, and CD4, CD5, CD8 T-cell specificities. We conclude that the surface of cultured LCs undergoes selective changes in culture. As a result, the cells are rich in Ia and H-2 and have detectable C3bi receptors, but have little or no LFA-1, Ti, CD4, 5, and 8, 33D1, 2.4G2, F4/80, and B220 antigens.