The first national survey of resistance to newer beta-lactams in nosocomial populations of Enterobacteriaceae in Poland was performed. The study covered all nonrepetitive enterobacterial isolates cultured from specimens from inpatients in 13 regional secondary-care hospitals from November 2003 to January 2004. Among 2,388 isolates, the predominant species was Escherichia coli (59.6%), followed by Proteus mirabilis (14.5%) and Klebsiella spp. (8.5%). The frequency of extended-spectrum beta-lactamases (ESBLs) was very high, with ESBLs present in 11.1% of all isolates and 40.4% of Klebsiella pneumoniae isolates, the latter value greatly exceeding that for E. coli (2.5%). The contribution of outbreak isolates was significant, resulting, for example, in a particularly high rate of ESBL producers among Serratia marcescens isolates (70.8%). The pool of ESBL types was overwhelmingly dominated (81.7%) by CTX-M-like beta-lactamases CTX-M-3 (80.6%) and CTX-M-15, with SHV types (17.5%; SHV-2, SHV-5, and SHV-12) and sporadic TEM-like enzymes (0.7%; TEM-19 and TEM-48) being the next most frequent. Acquired AmpC-type cephalosporinases were observed exclusively in P. mirabilis, in 20.5% of the isolates of this species (compared with the frequency of ESBL producers of 11.5% of P. mirabilis isolates). All these cephalosporinases (CMY-12, CMY-15, and a novel variant, CMY-38) originated from Citrobacter freundii. Four isolates of E. coli (two isolates), K. pneumoniae (one isolate), and P. mirabilis (one isolate) produced class A inhibitor-resistant beta-lactamases (TEM-30, TEM-32, TEM-37, and SHV-49), being the first of such producers identified in Poland. The survey documented both specific and more global characteristics of the epidemiology of the beta-lactamase-mediated resistance in enterobacteria from Polish hospitals and demonstrated that the ESBL frequency has reached an alarming level.