HL‐60 is a human promyelocytic cell line that differentiates along the granulocytic pathway when incubated with dimethylsulfoxide and along the monocytic pathway when incubated with 1,25‐(OH)2D3. We compared arachidonic acid metabolism in undifferentiated, DMSO‐differentiated, and 1,25‐(OH)2D3‐differentiated cells. DMSO‐ and 1,25‐(OH)2D3‐differentiated cells metabolized exogenous arachidonic acid to both cyclo‐oxygenase products (predominantly thromboxane B2 and prostaglandin E2) and 5‐lipoxygenase products, including leukotriene B4. Undifferentiated cells produce these metabolites in much smaller amounts. DMSO‐differentiated cells released a large percentage of phospholipid‐bound arachidonic acid in response to stimulation with the ionophore A23187, zymosan, or formylmethionylleucylphenylalanine (FMLP). DMSO‐differentiated cells stimulated with A23187 converted released arachidonate to LTB4 and TxB2. In contrast, 1,25‐(OH)2D3‐differentiated cells released a smaller percentage of phospholipid‐bound arachidonate in response to stimuli, and undifferentiated cells released none at all. The three cell types (undifferentiated, DMSO‐differentiated, and 1,25‐(OH)2D3‐differentiated) were homogenized and the 10,000 × g supernatant incubated with [14C]arachidonic acid. The supernatants from the homogenates of the DMSO‐ and 1,25‐(OH)2D3‐differentiated cells metabolized [14C]arachidonic acid to cyclooxygenase and lipoxygenase products, but the supernatant from the homogenate of undifferentiated cells did not. These data indicate that differentiation of HL‐60 cells with DMSO or 1,25‐(OH)2D3 induces cyclooxygenase and 5‐lipoxygenase and induces mechanisms for the release of arachidonate from phospholipids by soluble and particulate stimuli.