To study the effect of mononuclear cell differentiation on metalloproteinase production, the human monocytic cell lines U937 and THP-1 were exposed to two well known differentiating agents, the phorbol esters (phorbol myristate acetate (PMA)) and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). With U937 cells, PMA-induced differentiation increased the production of both interstitial collagenase and 92-kDa gelatinase, whereas exposure to 1,25-(OH)2D3 induced full interstitial collagenase expression in the absence of any detectable 92-kDa gelatinase production. In fact, when U937 cells were differentiated with PMA and then exposed to vitamin D3, the hormone actually suppressed phorbol-induced 92-kDa gelatinase biosynthesis. With THP-1 cells, PMA also induced the production of 92-kDa gelatinase fully, but unlike U937 cells, the combination of PMA and 1,25-(OH)2D3 was required for substantial interstitial collagenase biosynthesis. As with U937 cells, the addition of 1,25-(OH)2D3 to PMA-differentiated THP-1 cells caused a dose-dependent inhibition of 92-kDa gelatinase production. Northern hybridizations demonstrated that both phorbol esters and vitamin D3 act on monocytic cell lines at a pretranslational level. To determine whether metalloproteinase biosynthesis in normal differentiated mononuclear phagocytes was also modified by 1,25-(OH)2D3, human blood monocytes and alveolar macrophages were exposed to this hormone. In both cell types, basal and Staphylococcal-stimulated 92-kDa gelatinase production was markedly inhibited by 1,25-(OH)2D3. In contrast, interstitial collagenase production was completely unaffected by the hormone. In summary, the two major metalloproteinases produced by monocytic cells are regulated via distinct molecular pathways by the action of PMA and 1,25-(OH)2D3. Furthermore, vitamin D3 completely dissociates the production of 92-kDa gelatinase and interstitial collagenase in human mononuclear phagocytes.