Clinical isolates of Trypanosoma brucei rhodesiense, which were resistant to arsenical drugs in murine infections, were examined for resistance in vitro. A rapid lysis assay was developed which was able to predict in vivo sensitivity to melarsoprol (Mel B, Arsobal) and melarsen oxide. The assay was based on the finding that long slender bloodforms of drug-sensitive isolates would lyse in the presence of arsenicals upon incubation in heat-inactivated fetal bovine serum. On the basis of plots of decrease in the absorbance of trypanosome suspensions vs time of incubation with drug, L50 values, reflecting the drug concentration necessary for lysis of 50% of the cells within 30 min. were calculated for five strains. These values ranged from less than 30 microM for arsenical-sensitive strains to greater than 75 microM in proven arsenic refractory isolates. Calcium was essential for lysis, and the presence of the Ca2+ chelator EGTA (10 mM) in serum delayed lysis of sensitive strains. Ca2+ channel antagonists (Verapamil, Diltiazem), however, did not enhance lysis of refractory isolates when used at 20 to 30 microM. Intracellular concentrations of reduced trypanothione, the apparent target of arsenicals, were similar for all isolates, approximately 1.02 +/- 0.28 nmol/10(8) cells, as detected by monobromobimane derivitization and HPLC analysis. Uptake of melarsen oxide was found to be reduced in arsenical refractory strains. Uptake was judged by reduction of free reduced trypanothione as a result of formation of the trypanothione-arsenic complex Mel T. Little change was found in arsenical-resistant strains, but sensitive strains had 50 to 70% reductions in trypanothione levels after incubation with a low (1 microM) level of melarsen oxide.