Experimental factors and determinants of the protein binding of enoximone (a new cardiotonic agent) were investigated in human serum from healthy, drug-free subjects using a rapid ultrafiltration method; these factors and determinants included nonspecific binding to the apparatus, ultrafiltrate volume, temperature, serum pH, enoximone serum concentration, and enoximone sulfoxide (metabolite) concentration. It was demonstrated from mass balance experiments that nonspecific binding to the apparatus did not occur. Within the range investigated, ultrafiltrate volume did not affect the binding result. However, serum pH and temperature were critical variables. At pH 7.4 and 37 degrees C, enoximone serum binding occurred to the extent of approximately 70%; over the therapeutic serum concentration range, this binding was concentration independent. Experiments with purified albumin solutions indicated that much of the serum binding could be accounted for by albumin. At concentrations exceeding those observed clinically, enoximone sulfoxide did not affect enoximone serum binding. In another experiment, enoximone binding to serum was compared with that from plasma containing either heparin or disodium EDTA. There were essentially no differences. Enoximone sulfoxide serum protein binding was also investigated in serum from healthy, drug-free human subjects; binding occurred to the extent of approximately 5%.