A superfusion bioassay for platelet-activating factor is described using various types of tissues. By washing the tissue with 0.1-0.5% bovine serum albumin for 2-3 min after each addition of platelet-activating factor, desensitization did not develop in most tissues studied. Because of the ability to apply a sample directly onto an assay tissue with negligible dilution, this bioassay can detect smaller amounts of platelet-activating factor than those previously reported in which an organ bath was utilized. The ascending colon of the rat and dog appeared to be the most sensitive of the tissues tested, with a limited of detectability in the range of 100-500 fg. Repeated additions of platelet-activating factor could be made for up to 4 h without desensitization. Release of platelet-activating factor from samples of rat stomach was measured using the superfusion bioassay and a platelet aggregation bioassay. There was a significant correlation (r = 0.96; p less than 0.01) between the values obtained using the two assay systems. Thus, the sensitivity, the reproducibility, and the inexpensive nature of this bioassay make it an attractive alternative to existing bioassays for platelet-activating factor.