Mutants affecting regulation of thiamine biosynthesis from its pyrimidine and thiazole moieties were isolated from Escherichia coli K12 as resistant strains to growth inhibition by pyrithiamine, an antimetabolite of thiamine. The mutants (PT-R1 and PT-R3) have 3-fold higher cellular thiamine content than the parent strain. Among four enzymes involved in thiamine synthesis from its pyrimidine and thiazole moieties, the mutants contain derepressed levels of hydroxyethyl-thiazole kinasc [EC 2.7.1.50] and thiaminephosphate pyrophosphorylase [EC 2.5.1.3] and repressed levels of hydroxymethylpyrimidine kinase [EC 2.7.1.49] and phosphohydroxymethylpyrimidine kinase [EC 2.7.4.7]. The former two enzymes are not repressed by the end-product, thiamine, and the latter two enzymes can be derepressed by adenine that lowers the cellular thiamine content. Evidence is presented to indicate that these mutants are in the same class of mutation with only difference in the extent of the repressed level of phosphohydroxymethylpyrimidine kinase. By conjugation tests the chromosomal position corresponding to the mutation in PT-R1 was found to link to the arg and met loci, and probably very close to the thi locus. The mutants might be similar to operator constitutive mutants studied in other systems.