The present study describes comparative bioavailability of rifampicin (RIF) after administration of a single component RIF (450 mg) capsule and rifampicin-isoniazid (RIF-INH) (450+300 mg) fixed dose combination (FDC) capsule formulations. Six healthy male volunteers participated in a single dose, two treatment, two period, cross-over study. A sensitive, specific and accurate HPTLC method was developed, validated and employed for estimation of RIF and its major active metabolite, 25-Desacetylrifampicin (25-DAR) levels, in urine. Using the urinary excretion data various pharmacokinetic parameters: AUC(0-24), AUC(0-infinity), cumulative amount excreted in 24 h, peak excretion rate, etc. for both RIF and 25-DAR were calculated and compared statistically (ANOVA, 90% confidence interval for ratio). Significant decrease in the bioavailability ( approximately 32% as RIF and approximately 28% as 25-DAR) of RIF from FDC capsules was observed. The present bioavailability study confirms our serious doubts about the stability of RIF in presence of INH in acidic environment of stomach, which probably is the main factor responsible for the reduced bioavailability of RIF from RIF-INH combination formulations. This study underlines the fact that there is an urgent need to reconsider the formulation of the FDC product in order to minimize or avoid the decomposition of RIF in gastrointestinal tract.