The metabolism of arachidonic acid has been studied using hamster trachea in short-term organ culture. To study endogenous substrate utilization, tissue lipids were labeled with [3H]-arachidonic acid, whereas exogenous substrate turnover was assessed by the addition of 100 microM [14C]-arachidonic acid to the medium. Both exogenous and endogenous arachidonate were converted primarily to 6-keto-PGF1 alpha and PGE2, with varying amounts of an unidentified non-polar product noted. Production of the prostanoids increased steadily with time up to 24 hours. No significant generation of lipoxygenase products was found. Release of incorporated labeled arachidonic acid was nearly linear with time, resulting in the transfer of about 10% of the total label into the medium after 24 hours. About 3% of the total label was converted to prostaglandins. In the presence of 10 microM A23187, release of label was increased by only 25 to 60% relative to the control. Analysis of labeled compounds in the medium showed that this increase resulted from increased release of unchanged arachidonic acid, and that the yield of oxygenated products was the same as from the control incubations.