Some clones of the human histiocytic lymphoma line, U-937, were induced to differentiate into monocyte-like cells with loss of plating efficiency in agar by incubation with 0.1 to 10 nM 1 alpha,25-dihydroxycholecalciferol [1,25(OH)2D3]. At 1 nM, 40% of the cells of one sensitive clone exhibited differentiation after 2 days of incubation judging from assays for phagocytosis and capacity to reduce nitroblue tetrazolium. Induction appeared to occur by binding of the cholecalciferol to a specific cytoplasmic and/or nuclear receptor for 1,25(OH)2D3. However, the presence of this receptor was not sufficient for differentiation, since one clone which contained the receptor did not respond with differentiation upon addition of 1,25(OH)2D3. Differentiation induction did not require DNA synthesis but was blocked by agents which inhibit RNA or protein synthesis. It was also blocked by the calcium ionophore A 23187. A synergistic inducing effect was seen between 1,25(OH)2D3 and retinoic acid. In addition, the U-937 cells could be primed by a short incubation with 1,25(OH)2D3 to respond, with maturation, to the addition of agents which increase the intracellular level of cyclic adenosine 3':5'-monophosphate, such as prostaglandin E2, cholera toxin, and N6,O2'-dibutyryl adenosine 3':5'-monophosphate and which alone did not induce differentiation. Priming does not depend on the normal rate of RNA or protein synthesis, since it was not significantly inhibited by actinomycin D, cordycepin, or cycloheximide. It remains to be determined if unoccupied receptors for 1,25(OH)2D3 are present in fresh leukemia cells and if such cells can sometimes be induced to differentiate upon addition of cholecalciferol.