TEM-1, OXA-1, SHV-1, and related beta-lactamases in fecal isolates from 953 infants in 22 Swedish neonatal intensive care units were studied by DNA hybridization. TEM-1- and OXA-1-positive isolates were always Escherichia coli and represented 86 and 8%, respectively, of the ampicillin-resistant isolates of this species. SHV-1 was found in 16% of the Klebsiella sp. (mainly Klebsiella pneumoniae) isolates. TEM-1 and SHV-1 occurred in 14 and 16 units and in up to 64 and 26% of the neonates, respectively. On average, two to four different biochemical phenotypes per species per ward were positive for each beta-lactamase. All but 1 of the 33 E. coli phenotypes found to be TEM-1 positive were uniformly positive for the beta-lactamase gene, whereas some of the phenotypes found to be positive for OXA-1 (2 of 3) and SHV-1 (6 of 70) were occasionally negative for the respective genes. The occurrence of the three beta-lactamases studied tended to be associated with local ampicillin usage (correlation coefficient, 0.31 to 0.39; P greater than 0.05). Of the neonates receiving ampicillin, 30% carried TEM-1-positive E. coli, compared with 13% for cephalosporin-treated neonates and 15% for untreated neonates (P less than or equal to 0.001). The corresponding rates for SHV-1 in Klebsiella spp. were 18, 13, and 9% (P less than or equal to 0.01). Ampicillin is thus a significant risk factor for the maintenance of the most prevalent gram-negative plasmid-mediated beta-lactamases in hospitalized neonates.