Megakaryocytes isolated from guinea pigs produced thromboxane (assayed by radioimmunoassay as thromboxane B2) in response to the platelet aggregatory stimuli arachidonic acid, thrombin, and the calcium ionophore A23187. The relative responses to these stimuli were similar in megakaryocytes and platelets from the same animals. When the megakaryocytes were maintained in short-term in vitro culture, all three stimuli still elicited thromboxane production. Following overnight in vitro culture, thromboxane production in response to thrombin decreased, overall, to under 60% of control values, increased approximately threefold in response to A23187, but did not show any alteration in response to arachidonic acid. Requirements for calcium were virtually unchanged. These results demonstrate that megakaryocytes contain all of the pathways needed for arachidonic acid mobilization from phospholipids in response to thrombin or A23187 and conversion of that arachidonate to thromboxane. These pathways are retained by the cells in short-term in vitro culture.