A sensitive method for determination of incorporation of non-radiolabeled 5-fluorouracil (FUra) into RNA (F-RNA) of tumor tissue samples was established and was shown to be applicable to clinical materials. RNA fractions containing incorporated FUra were extracted from perchloric acid precipitates of tissue sonicates by KOH hydrolysis. FUra in RNA fractions was released by HCl hydrolysis at 100 degrees C and its levels were determined by GC-MS. The sensitivity of this method was 100 fold higher than that of HPLC method. F-RNA levels were dose dependent and correlated well with antitumor efficacy of the drugs. F-RNA levels in FUra resistant murine tumors were significantly lower than that in parent tumors despite of the lack of difference in FUra concentrations in tumor tissue. Based on these results, it is suggested that measurement of F-RNA levels together with the determination of FUra concentration and the inhibition rate of thymidylate synthase should be considered as a good parameters of the evaluation of antitumor efficacy of FUra and its analogs both in experimental and clinical settings.