Two physically and genetically distinct forms of ribosephosphate isomerase have been identified in Escherichia coli K12. The constitutive ribosephosphate isomerase A has a Km for ribose 5-phosphate (4.4 +/- 0.5 mM) six times greater than that of the inducible ribosephosphate isomerase B (0.83 +/- 0.13 mM). Treatment of the enzymes with 1.25 mM iodoacetate resulted in 100% loss of activity for ribosephosphate isomerase B, whereas ribosephosphate isomerase A was unaffected. Various cellular metabolites were tested and found to be without significant effect on either enzyme. The two enzymes could be separated by filtration on Sephadex G75 superfine and their apparent molecular weights were 45000 for ribosephosphate isomerase A and 32000-34000 for ribosephosphate isomerase B. Under certain conditions the two enzymes showed different patterns of heat inactivation but the results with ribosephosphate isomerase A varied in an unusual way with the protein concentration. Ribosephosphate isomerase B was formed inducibly in a mutant lacking ribosephosphate isomerase A but there was no evidence for the production of ribosephosphate isomerase B in wild-type cells. The formation of ribosephosphate isomerase B was not a consequence of the ribosephosphate isomerase B mutation, since strains could be constructed which formed both enzymes constitutively in the anticipated amounts. The ribosephosphate isomerase formed by a secondary mutant obtained from a ribosephosphate-isomerase-A-negative strain was identified as ribosephosphate isomerase B on the basis of its Km, elution profile from Sephadex G75, inhibition of iodoacetate, and heat inactivation. The ribosephosphate isomerases of another Escherichia coli K12 strain, X289, were investigated, since their properties were reported to be different from many of these described here for ribosephosphate isomerases A and B. In our hands strain X289 contained two ribosephosphate isomerases apparently identical to ribosephosphate isomerases A and B. The evidence to date suggests that ribosephosphate isomerase A catalyses the formation of ribose 5-phosphate from ribulose 5-phosphate and also participates in the reverse reaction during ribose and adenosine catabolism. The normal physiological role of the inducible ribosephosphate isomerase B is still uncertain.