We have studied resistance to sulfadoxine-pyrimethamine (S/P) in the rodent malaria parasite Plasmodium chabaudi. A stable S/P-resistant mutant, AS(50S/P), was selected by drug treatment of a clone, AS(PYR), already resistant to pyrimethamine. The sequences of the P. chabaudi dhfr and dhps genes were obtained and found to be identical in AS(50S/P) and AS(PYR), showing that resistance to S/P in AS(50S/P) was not due to additional mutations in either gene. AS(50S/P) was crossed with a drug-sensitive clone, AJ, and 16 independent recombinant progeny were obtained. These clones were phenotyped for their susceptibility to S/P and to sulfadoxine and pyrimethamine separately. Pyrimethamine resistance was invariably associated with S/P resistance, but no correlation was found between resistance to S/P and resistance to sulfadoxine. Quantitative trait locus analysis of the progeny with 31 chromosome-specific markers showed that mutant P. chabaudi dhfr, or one or more genes closely linked to it, was a major determinant of S/P resistance. In addition, the inheritance of genes on chromosomes 5 and 13 from the sensitive parent appeared to contribute to the level of resistance observed. These results demonstrate that the S/P resistance of the AS(50S/P) mutant of P. chabaudi does not involve mutation in dhps and is not due simply to a combination of two genes determining resistance to pyrimethamine and sulfadoxine separately.