Mycobacterium microti incorporated a wide range of exogenously supplied pyrimidines into its nucleic acids. M. avium incorporated a relatively narrow range of pyrimidines but both M. avium and M. microti when recovered after growth in vivo incorporated a slightly wider range of pyrimidines than the same strains grown in vitro. M. microti and M. leprae could not take up uridine nucleotides directly but could utilize the pyrimidines by hydrolysing them to uridine and then taking up the uridine. Pyrimidine biosynthesis, judged by the ability to incorporate carbon from CO2 or aspartate into pyrimidines was readily detected in non-growing suspensions of M. microti and M. avium harvested from Dubos medium, which does not contain pyrimidines. The biosynthetic activity was diminished in mycobacteria grown in vivo when there is likely to be a source of pyrimidines which they might use. Relative activities for pyrimidine biosynthesis de novo in M. microti were 100 for cells isolated from Dubos medium, 6 for cells isolated from Dubos medium containing the pyrimidine cytidine and 11 from cells recovered after growth in mice. In contrast, relative activities for a scavenging reaction, uracil incorporation, were 100, 71 and 59, respectively. Three key enzymes in the pathway of pyrimidine biosynthesis de novo were detected in M. microti and M. avium. Two, dihydroorotate synthase and orotate phosphoribosyltransferase appeared to be constitutive in M. microti and M. avium. Aspartate transcarbamoylase activity was higher in these mycobacteria grown in vivo than in Dubos medium but it was repressed in M. microti or M. avium grown in Dubos medium in the presence of 50 microM-pyrimidine. Aspartate transcarbamoylase was strongly inhibited by the feedback inhibitors ATP, CTP and UTP. Enzymes for scavenging pyrimidines were detected at low specific activities in all mycobacteria studied. Activities of phosphoribosyltransferases, enzymes that convert bases directly to nucleotides, were not related to the ability of intact mycobacteria to take up pyrimidine bases while activities of pyrimidine nucleoside kinases were generally related to the ability of intact mycobacteria to take up nucleosides. Phosphoribosyltransferase activity for uracil, cytosine, orotic acid and--in organisms grown in Dubos medium with 50 microM-uridine-thymine, as well as kinases for uridine, deoxyuridine, cytidine and thymidine were detected in M. microti. However, M. avium only contained uracil and orotate phosphoribosyltransferase, uridine, cytidine and thymidine kinase, and additionally deoxyuridine kinase when grown axenically with 50 microM-uracil, reflecting its more limited abilities in pyrimidine scavenging.