A simple high-performance liquid chromatographic method for determination of rifapentine, a cyclopentyl semisynthetic analogue of rifamycin belonging to the class of piperazinyl hydrazone derivatives of 3-formylrifamycin SV, and its metabolite, 25-desacetylrifapentine, in human plasma was developed using direct injection of the sample onto a Supelco LC HISEP column. The mean recovery was 100.3% for rifapentine and 99.7% for the metabolite and the precision of the assays was 3% and 7%, respectively. The limit of determination was 0.2 micrograms/ml and the method was validated for concentrations up to 64 micrograms/ml for rifapentine and 32 micrograms/ml for the metabolite. The results correlated well with those of the microbiological assay with Sarcina lutea as test organism.