The in vitro activity of BL-S640 (cefatrizine) was determined against 674 recent clinical isolates of Staphylococcus aureus and Enterobacteriaceae. Activity against S. aureus was less than that of cephapirin, cephalothin, and cefazolin, but greater than that of cephalexin. Activity against gram-negative isolates was variable: BL-S640 was slightly less potent than cefazolin against Escherichia coli and Klebsiella, but more active than the other compounds. As for the more resistant gram-negative genera, BL-S640 was significantly superior to the control cephalosporins. The effect of inoculum size on the antibacterial activity was moderate for most organisms except Enterobacter, Providencia stuartii, and indole-positive Proteus, the median minimal inhibitory concentrations of which were 6 to 27 times lower when determined with a 10-4-diluted culture compared with the undiluted one. The stability in aqueous solution at 37 C was remarkably high at the lower pH values, but low at the neutral point.