In order to determine the contribution of thymidine (dThd) salvage to intrinsic resistance to antimetabolites (5-fluoropyrimidines, antifolates) in the human colon adenocarcinoma xenograft, H X GC3, a subline deficient in thymidine kinase has been developed. A cell line (GC3/M) was established in continuous culture that demonstrated a karyotype identical to that of the stem line of H X GC3 (46,X, - Y + 12). After inoculation of GC3/M cells into immune-deprived CBA/CaJ mice, the H X GC3/M xenografts retained histological, histochemical, and growth characteristics of the H X GC3 xenograft. To develop a line deficient in dThd salvage, GC3/M cells were selected with BrdUrd (100 micrograms/ml). Three clones characterized were unable to proliferate in HAT medium, and were deficient (less than 10% control) in the cytosolic form of thymidine kinase. Activities of dThd phosphorylase and dTMP synthase were unchanged from parental GC3/M cells. Of the three clones inoculated into mice, GC3/M TK- 100 C3 was tumorigenic, the xenografts demonstrating histological and growth characteristics similar to H X GC3. The in vivo activity of the cytosolic form of dThd kinase was 3.5% of that in H X GC3 xenografts. Incorporation in vivo of [methyl-3H]dThd into acid insoluble material was 14% of that in H X GC3 tumors. Autoradiographs prepared from these tumors demonstrated that incorporation of radiolabel into nuclei occurred only in stromal cells derived from the host. It is anticipated that H X GC3/M TK- 100 C3 will be a line valuable for determining the role of dThd salvage in intrinsic resistance to 5-fluoropyrimidines or antifolates in human colon adenocarcinomas growing as xenografts and also the relevance of dTMp synthase as a target for antimetabolites in this histiotype.