We have determined the nucleotide sequence of the ereB gene of plasmid pIP1527 which confers high-level resistance to erythromycin by inactivation in Escherichia coli. The open reading frame of the ereB gene, 1257-bp, was defined by initiation and termination codons and by cloning in vitro. The corresponding protein has a calculated Mr of 48,118 in close agreement with a previous estimation, 51,000, by electrophoresis of minicell extracts in SDS-polyacrylamide gels. The structure of the modified erythromycin was determined by physico-chemical techniques including mass spectrometry, infrared spectrophotometry and 13C nuclear magnetic resonance. The data obtained indicated that like ereA (Ounissi and Courvalin, 1985) ereB encodes an erythromycin esterase. Comparison of the amino acid sequences of the two isozymes did not reveal any statistically significant homology. Analysis of the nucleotide sequence of the ereB gene suggests that this resistance determinant should be exogenous to E. coli.