cDNAs which encode the rat testis and pancreatic islet mitochondrial glycerol phosphate dehydrogenase (mGPD) (EC 1.1.99.5), the key enzyme of the glycerol phosphate shuttle, were recently cloned and sequenced and found to contain calmodulin-like calcium-binding sequences, thus explaining the widely observed calcium activation of the enzyme from many tissues of higher eukaryotes. mGPD activity and protein, as judged from Western analysis, appear to be most abundant in testis and pancreatic islets in the rat. mGPD is known to be located within the inner mitochondrial membrane. At a physiologic concentration of glycerol phosphate (75 microM), half maximal activity of Triton X-100-solubilized testis mGPD was seen in the presence of 0.1-0.25 microM free calcium. Calcium (10(-6)-10(-5) M) lowered the Km of mGPD from 2.5 mM glycerol phosphate (islet mGPD) and 3.2 mM glycerol phosphate (testis mGPD) to 0.4 mM glycerol phosphate. Calcium activation of mGPD from both testis and islets was not prevented by calmodulin inhibitors, which is consistent with mGPD possessing regions that can mediate its own activation by calcium. 45Calcium overlay experiments, in which proteins were separated by SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and probed with 45Ca, showed that mGPD is a major calcium-binding protein in testis mitochondrial membranes. A hydropathy plot suggested that the mature mGPD protein has three transmembrane helices. The first membrane-spanning region coincides with the FAD site and thus this site is placed within the membrane. The hydropathy analysis indicated that the calcium-binding region and the putative glycerol phosphate-binding site lie outside the membrane exposed to the cytosolic environment. This is consistent with earlier biochemical evidence which indicated that these sites are situated outside the membrane (M. Klingenberg, Eur. J. Biochem. 13, 247-252, 1970). This suggests that cytosolic calcium can regulate mGPD activity. Since simultaneous oscillations in electrical activity, cytosolic calcium, glycolysis, and insulin release occur in the pancreatic beta cell, mGPD activity might also fluctuate and allow the glycerol phosphate shuttle to participate in glycolytic oscillations.