To study the national distribution of beta-lactam resistance patterns and mechanisms among Escherichia coli organisms isolated in U.S. hospitals, 652 ampicillin-resistant (Am(r)) or ampicillin-intermediate (Ami) isolates were submitted to the Centers for Disease Control from March 1983 through July 1984 by nine hospitals participating in the National Nosocomial Infections Study. Among the isolates (most of which caused urinary tract infections), 78% were Am(r) and 22% were Ami by the interpretative criteria established by the National Committee for Clinical Laboratory Standards. Resistance to carboxypenicillins ranged from 73 to 74%, and that to acylureidopenicillins ranged from 43 to 66%. A total of 26% of the isolates were resistant to cephalothin, and 4% were resistant to cefazolin. Resistance to cefoxitin was 1%, while resistances to cefuroxime and cefamandole were 2 and 7%, respectively. With the exception of cefsulodin (98% resistant) and cefoperazone (1% resistant), there was no resistance to newer cephalosporins or aztreonam. In general, only minor differences in the incidence of resistance to beta-lactam antibiotics were noted in hospital-acquired versus non-hospital-acquired isolates as well as among isolates from various regions of the United States. TEM beta-lactamases were produced by 87% of the 237 Am(r) isolates examined. By our methods, OXA and chromosomal (type I) beta-lactamases were detected in 2 and 28 isolates, respectively, and plasmid-mediated extended-spectrum cephalosporinases were detected in none of the isolates. Disk substrate and clavulanic acid inhibition assays revealed that TEM beta-lactamase conferred Am(r) and resistance to carboxypenicillins, acylureidopenicillins, cephalothin, cefamandole, cefsulodin, and cefoperazone. A total of 391 isolates were screened for plasmids, and 259 isolates were examined by DNA hybridization with a TEM probe. Among 462 plasmids probed, 129 plasmids, ranging from 4 to 140 megadaltons, harbored TEM sequences. Although beta-lactam resistance in clinical isolates of E. coli is predominantly mediated by TEM beta-lactamase, the diverse spectrum of resistance appears to be related to additional strain=dependent factors.