A well-known problem of anti-tuberculosis fixed-dose combination (FDC) products containing rifampicin (R) and isoniazid (H) is the fall in bioavailability, in particular of R, when two or more drugs are present together. The same has been ascribed to hydrolysis of R to 3-formylrifamycin (3-RIF) under stomach acid conditions and reaction of the latter with H to form isonicotinyl hydrazone (HYD). The objective of present study was to explore whether the same reaction occurred when H was present along with rifapentine (Rp), a newer long acting rifamycin, which is structurally similar to R. Clinical trials are currently undergoing for co-administration of Rp with H in patients who had completed 2 months of standard chemotherapy. For the purpose, first a validated HPLC method was developed for the separation of Rp and H, and the same was used for the study of interaction between the two drugs. Like R, Rp was also found to convert to 3-RIF in acid conditions, which reacted further with H to form HYD. The pH-rate profile was also similar in shape to that established with the combination of R and H; maximum decomposition occurred at pH 2, where Rp loss was to an extent of approximately 30%, while corresponding decomposition of H was approximately 9%. These values were similar to those reported for the combination of R (approximately 33%) and H (approximately 10%). Hence, the study suggests that co-administration of Rp and H should be avoided, like in case of R and H, and the two drugs should not be formulated directly into a single dosage form.