Genes coding for production of the K1 polysaccharide capsule of Escherichia coli have been cloned. Complementation, insertion, and deletion analyses were used to localize the K1 genes and demonstrated that a minimum of 9 kilobases of DNA split into at least two gene blocks is involved in synthesis and assembly of the capsule. One of the gene blocks is responsible for biosynthesis of the polysaccharide, and the other is responsible for extracellular appearance of capsular material. Using cloned K1 genes as probes in Southern blot experiments, we detected homology to DNA from strains of E. coli capsular types K92, K7, and K100. In contrast, no homology was apparent between K1 genes and DNA from meningococcus group B, although the K1 and group B capsules are chemically and immunologically identical.