Biological characterization of a human colon adenocarcinoma cell line deficient in thymidylate synthase (TS-) is described. The clone, designated TS-C1/C1, was derived from the parental line GC3/C1 by selection in medium containing aminopterin, thymidine (dThd), and low concentrations of 5-formyltetrahydrofolate (5-CHO-H4PteGlu), and was subsequently reselected by single-step cloning in 500 microM methotrexate in the presence of dThd. This clone retained its TS- phenotype, was highly resistant to methotrexate (greater than 100,000-fold), and remained tumorigenic in mice (P.J. Houghton, et al., Proc. Natl. Acad. Sci. USA, 86: 1377-1381, 1989). In studies reported, it is shown that high levels of exogenous folate can support the growth of the TS- C1/C1 clone in the absence of dThd. Activation of dTMP biosynthesis de novo was demonstrated within 6 h of exposing cells to 20 microM [6R,S]5-CHO-H4PteGlu, and greater than or equal to 80% of activity was lost within 24 h of removing this folate from the medium. The labeling index was determined by autoradiographic techniques using [6-3H]2'-deoxyuridine. None of the greater than 6,000 cells radiolabeled in the absence of [6R,S]5-CHO-H4PteGlu, whereas 33.5% labeled in the presence of 20 microM exogenous folate. Relative to the parental (TS+) clone, there was a greater than 87,500-, an 8,182-, and a 425-fold higher requirement for 5-methyltetrahydrofolate ([6R,S]5-CH3-H4PteGlu), PteGlu, and [6R,S]5-CH3-H4PteGlu to support 50% maximal colony formation in the absence of dThd. Quantitative analysis of the combined pools of 5,10-methylenetetrahydrofolate (CH2-H4PteGlun) and H4PteGlun showed that parental GC3/C1 cells had higher endogenous folate pools compared to TS-C1/C1 cells [168 +/- 40 (SD) and 10.9 +/- 0.3 fmol/10(6) cells, respectively]. Qualitatively the distribution of polyglutamate species and their redistribution in cells exposed to 20 microM [6R,S]5-CHO-H4PteGlu were similar in the two lines. Analysis of pools in a second, independently derived, TS- clone (TS-C3/C3, a transcription-negative mutant) demonstrated undetectable levels of CH2-H4PteGlun and H4PteGlun. This line cannot be rescued by exogenous folate. The data thus suggest that deletion of dTMP synthase activity may cause redistribution of reduced folate pools. In cytosolic extracts from parental GC3/C1 (TS+) cells, [6R]CH2-H4PteGlu1 acted as a cofactor in the release of 3H2O from [5-3H]dUMP, whereas no activity was detected in cytosols from TS-C1/C1. In contrast dTMP synthase activity was detected in cytosols from TS- C1/C1 cells in the presence of [6R]CH2-H4PteGlu5.(ABSTRACT TRUNCATED AT 400 WORDS)