The panB gene from Escherichia coli, encoding the first enzyme of the pantothenate biosynthesis pathway, ketopantoate hydroxymethyltransferase (KPHMT), has been isolated by functional complementation of a panB mutant strain with an E. coli genomic library. The gene is 792 bp long, encoding a protein of 264 amino acids with a predicted M(r) of 28,179. The identity of the gene product as ketopantoate hydroxymethyltransferase was confirmed by purification of the enzyme protein, which was overexpressed approximately 50-fold in the mutant harboring the gene on a high-copy-number plasmid. The N-terminal amino acid sequence of the purified protein was found to be identical to that predicted from the gene sequence, as was its mass, determined by electrospray mass spectrometry. Upstream of the panB gene is an incomplete open reading frame encoding a protein of 220 amino acids, which shares sequence similarity to fimbrial precursor proteins from other bacteria. Northern (RNA) analysis showed that the panB gene is likely to be cotranscribed with at least one other gene but that this is not the putative fimbrial protein, since no transcripts for this gene could be detected.