The effects of 1,25-dihydroxy vitamin D3 and gamma interferon on the phenotypic changes associated with monocyte maturation in vitro were investigated. Human monocytes separated from peripheral blood mononuclear cell populations by adherence to plastic were cultured for 7 days on glass. Immunocytological analysis was performed on monolayers fixed at various times by using monoclonal antibodies specific for mature macrophages (RFD7), interdigitating (dendritic) cells (RFD1), and class II major histocompatibility complex antigen (RFDR1). Without any addition to the culture medium, proportions of these monocytes (normally RFD1 and RFD7 negative) developed either RFD1 positivity (23%) or RFD7 positivity (49%) over 7 days of culturing. The addition of gamma interferon to these cultures markedly reduced the proportion of RFD7-positive cells (less than 10%) but increased the proportion of RFD1-positive cells (40 to 60%). In contrast, 1,25-dihydroxy vitamin D3 reduced the expression of both RFD1 and RFD7. Both of these effects were dose dependent and required at least 3 days of contact with the cells. The possibility that RFD1- and RFD7-positive cells represent functionally distinct subsets makes these effects of significance in our understanding of the role of these mediators in controlling the immunocompetence of nonlymphoid accessory cell populations and in macrophage-associated antimicrobial activity.