Ochratoxin A (OTA), a mycotoxin widespread in cereals, occurs in polished rice that is consumed as cooked rice after washing and steaming. Cooking decreases OTA levels in food to varying extents, but little is known about how cooking changes the biological activity of this mycotoxin. We therefore evaluated the fate of OTA during rice cooking to determine the OTA residues and cytotoxic potential in vitro. Water-washed rice, ordinary cooked rice, and pressure-cooked rice were prepared from three polished rice lots naturally contaminated with OTA. Residual OTA in each sample was analyzed by high-performance liquid chromatography (HPLC), whereas in vitro cytotoxicity of OTA to C6 glioma cells, susceptible to low levels (nanograms per milliliter) of OTA, was used to confirm the chemical analysis. OTA concentration, as determined by HPLC analysis, in the cooked rice by both types of cookers was significantly lower than (59 to 75%) in the raw polished rice and water-washed rice. The cytotoxicity of the OTA that remained in the pressure-cooked rice from three lots was markedly decreased (approximately 20%, P < 0.05) when compared with other samples in respective lots. This confirms that cooking lowers OTA residues. Although washing polished rice with water had little effect on OTA levels, pressure steaming appeared to be the critical cooking step not only to reduce OTA residues in polished rice before reaching the consumer as the dietary staple of cooked rice, but also to diminish cytotoxicity of OTA.