Mouse peritoneal macrophages were incubated with varying amounts of heat-inactivated fetal calf serum (FCS) and treated with diazepam at the concentrations of 0.5 and 5.0 mug/ml. These macrophage preparations demonstrate that the capacity for synthesis and release of prostaglandins (PGs) E2, F2 alpha, 6-keto-PGF1 alpha and thromboxane TX(B2) is: (a) sensitive to the concentration of FCS in the culture medium, and (b) altered in the presence of diazepam. A radioimmunoassay for 6-keto-PGF1 alpha is also reported. The efficiency of extraction of PGE2 and 6-keto-PGF1 alpha from buffer or plasma samples was greatly improved when a petroleum ether/ether (1/1) solvent system was employed.