From 1980 to 1986 a regional survey was performed to investigate the susceptibility of β-lactam antibiotics in clinical isolates of Escherichia coli, Proteus mirabilis, Klebsieila pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa in 6 hospitals located in the southern part of the Netherlands. Each year 300 to 400 strains were tested. The susceptibility to representatives of the older and the newer β-lactam antibiotics was determined by a microbroth dilution assay using an inoculum size of 105 colony forming units (CFU)/ml in ISO-Sensitest broth (Oxoid). No significant differences in antibiotic sensitivity of the micro-organisms from the different hospitals were found. There was no evidence for an increase in antibiotic resistance of the strains during the sampling period. In contrast, there was a general tendency to an increase in antibiotic sensitivity.