A methylcholanthrene-induced rat sarcoma propagated both in vitro and in vivo was used to examine the usefulness of a rapid biochemical in situ assay that measures thymidylate synthase (TS) activity in whole cells to predict sensitivity and resistance to the folate antagonists methotrexate (MTX), 10-ethyl-10-deazaaminopterin (10-EDAM) and trimetrexate (TMTX). There was an excellent correlation between the effects of these drugs on inhibiting TS and cytotoxicity as measured by a clonogenic assay, when the exposure time was 4 hr and those when the rat sarcoma cell line was employed (p less than 0.001). When tumor-cell suspensions were prepared from the rat sarcoma propagated in vivo, they were less sensitive to these antifolates, but the relative effectiveness of the 3 antifolates was similar: TMTX much greater than 10-EDAM greater than MTX. As expected, continuous exposure (10 to 12 days) produced cytotoxicity at a much lower dose of these drugs. When the 3 drugs were tested for anti-tumor effectiveness in rats bearing this tumor, the tumor regression measured was best with TMTX; 10-EDAM was intermediate in effectiveness as compared with MTX. These results indicate that the in situ TS assay and clonogenic assay may be used to predict anti-tumor efficacy of antifolates in vivo in this rat sarcoma.