The kinetic interaction of cephalothin and cefamandole with human serum protein and of cephalothin with canine serum protein was studied with use of a rate-of-dialysis method. Binding of antibiotic to serum proteins was complete in less than 1 min and was immediately reversible. Human serum bound 76.9% of cephalothin and 73.7% of cefamandole at 10 microgram/ml, and canine serum bound 41.3% of cephalothin at 10 microgram/ml when this technique was used. Quantitative binding determined by ultracentrifugation was 78.5% for cephalothin and 80.1% for cefamandole in human serum and 45.6% for cephalothin and 32.5% for cefazolin in canine serum. There was no delayed binding of these antibiotics to either human or canine serum. In addition, a large number of antibiotic-binding sites appeared to be present on both human and canine serum protein when the antibiotic concentration was increased to 10(5) microgram/ml.