The operation of reductive pyrimidine catabolic and reutilization pathway in Tetrahymena pyriformis was investigated. Consistent with the proposed catabolic interconversions, radioactivity from [2,6-14C2]thymidine was recovered in respired CO2 30 min after its addition to the culture, whereas, consistent with the proposed anabolic interconversions, over 50% of the incorporated label was recovered in cellular macromolecules other than DNA 12 h after its addition. The chromatographic recovery of 14C radioactivity in monosaccharides from [2,6-14C2]thymidine as well as from [2-14C]methylmalonic acid, a key reutilization intermediate in this proposed pathway, further substantiated the operation of the required anabolic interconversions in this organism.