We evaluated the prevalence of impermeability as a mechanism associated with resistance against beta-lactam antibiotics in members of the family Enterobacteriaceae. During a 1-year period, 80 strains were selected from 3,110 routinely isolated strains according to their noticeable cross-resistance pattern to cephalosporins. They were tested for (i) outer membrane nonspecific porins involved in the entry of small hydrophilic molecules; (ii) the MICs of cefepime, cefotaxime, imipenem, and moxalactam; and (iii) beta-lactamase production. Immunological investigations using specific probes showed that 23 of 80 strains presented an alteration of the porin content, most of them expressing an additional resistance mechanism. The prevalence of this porin-deficient phenotype is especially high in Enterobacter aerogenes and concerns 6.4% of the clinical isolates.