ETHNOPHARMACOLOGICAL RELEVANCE: Zuojin Pill (ZJP) is a traditional Chinese medicine (TCM) formula composed of Coptidis Rhizoma and Euodiae Fructus in a ratio of 6:1 (w/w), which has been widely used for treating gastrointestinal disorders, especially stomach heat syndrome (SHS). However, the active alkaloids in ZJP showed low plasma exposure in rats following oral administration, which failed to explain their potent pharmacological effects, thereby limiting further mechanism studies. AIM OF THE STUDY: This study aimed to investigate the in vivo exposure and tissue distribution propensities of the active alkaloids in normal and SHS rats following oral administration of ZJP. MATERIAL AND METHODS: A rat model of SHS was induced by oral administration of chili pepper decoction and anhydrous ethanol. Then, the plasma and tissue pharmacokinetics of active alkaloids, including four protoberberine alkaloids (PBAs) and three indole alkaloids (IDAs), were investigated following oral administration of ZJP. Furthermore, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) was employed to characterize the spatial distribution of active alkaloids in the stomach and liver. Western blot and immunofluorescence were employed to evaluate the gastric mucosal barrier integrity. RESULTS: Based on the tissue-to-plasma partition coefficient (Kp) values, the in vivo exposure levels of berberine (BBR), palmatine (PAL), coptisine (COP), and dehydroevodiamine (DHE) were found to be higher in tissues than in plasma, indicating a distinct tissue distribution propensity. Each alkaloid displayed the highest exposure in the gastrointestinal tissues, due to local penetration facilitated by its direct contact with the mucosal lining. Pathological states reduced the overall exposure of PBAs in the gastric mucosa. In non-gastrointestinal tissues, most alkaloids, especially BBR and COP, exhibited a potent liver distribution propensity with minimal impact from pathological states. According to DESI-MSI results, PBAs showed high exposure in the damaged regions of gastric mucosa, which was attributed to mucosal barrier damage and enhanced permeability. In the liver, PBAs were primarily localized in the parenchyma surrounding the central vein and portal area. CONCLUSION: This study demonstrated the stomach and liver distribution propensity of the active alkaloids in ZJP, providing a scientific basis for these alkaloids as the pharmacodynamic material basis of ZJP against SHS from the perspective of drug exposure.