A gas chromatographic-mass spectrometric method for detecting 5-fluorouracil (5-FU) in serum at concentrations as low as 10 ng/ml was used to determine to what extent 5-FU was present in the serum of patients taking oral 5-fluorocytosine (5-FC). Preliminary studies in two patients and two healthy volunteers given an initial 2-g oral dose of 5-FC demonstrated sustained serum 5-FU levels (>100 ng/ml) during the 5 h after ingestion of drug. Pharmaceutical preparations of 5-FC used in these studies were shown to be insignificantly contaminated with 5-FU (<0.03%), suggesting in vivo conversion of 5-FC to 5-FU had occurred. Serum samples from seven patients with cryptococcal meningitis treated with amphotericin B and 5-FC were examined for 5-FU. Five of these patients had experienced hematological or other toxicity attributed to 5-FC at some time during the course of therapy. Of 41 serum samples, 20 were observed to have 5-FU levels greater than 1,000 ng/ml in the range observed with cancer chemotherapeutic doses of 5-FU known to be associated with hematological toxicity. It is concluded that conversion of 5-FC to 5-FU occurs in humans and furthermore that 5-FU may account for some of the toxicity observed with 5-FC.