The existence of the two crystalline forms of chlortetracycline hydrochloride (CTC-HCI) was confirmed by infrared spectroscopy and X-ray diffraction. From the dissolution studies with crystalline powder and compressed disk, an appreciable difference in the dissolution behavior in water was detected between the two forms. In order to determine the effect of polymorphism on the gastrointestinal absorption of CTC-HCl, blood plasma levels obtained in rabbits after intraduodenal administration and cumulative amounts excreted in human subjects after oral administration of the two forms were compared. The results indicated that polymorphic state of CTC-HCl significantly influences bioavailability of the CTC-HCl.