Cyclacillin was compared with cephalex-in and ampicillin for activity against various patho- gens in vitro and the corresponding infections they produce in mice. Cyclacillin was superior to cephalex- in in vitro and in vivo against penicillin-sensitive S. aureus and Strep, pyogenes. Against penicillin- ase-producing staphylococci, cyclacillin was clearly superior to cephalexin in vivo, while in vitro cephalexin was the more active of the two. Cyclacillin and ampicilin were about equally active in vivo against Strep, pyogenes, D. pneumoniae and penicillin-sensitive staphylococci, while in vitro cyclacillin was somewhat less active than ampicillin. Against penicillin-resistant staphylococci, cyclacillin was active while ampicillin was not. Against gram-negative organisms cyclacillin was less active both in vitro and in vivo than ampicillin. Faced with an increasing challenge by the penicillin-sensitive cocci (i. e., treatment of mice infected with 100 to 100,000 LD95), cyclacillin was clearly and consistently superior to ampicillin. The high order of therapeutic activity exhibited by this agent cannot be predicted from the in vitro susceptibility data. There is no satisfactory explanation for this disparity, which is known to occur also with some other antimicrobial agents. The present results confirm the need for careful consideration of all the relevant biologic parameters before extrapolating in vitro data to the clinical situation.