High levels of phosphocreatine, a compound known to serve as an intracellular energy reserve, were found in the fluid contained in seminal vesicle glands. The concentrations of phosphocreatine in the extracellular fluid in the mouse and rat were found to be 5.6 ± 1.6 and 2.2 ± 0.8 ,umol/g, respectively, which are higher than the intracellular levels reported for smooth muscles. The creatine concentrations in the seminal vesicular fluid from these two species were 22.8 ± 3.1 and 13.0 ± 5.3 ,umol/g, respectively. These creatine levels are approximately 100 and 65 times higher than the creatine levels in mammalian blood. Smaller amounts of ATP (phosphocreatine/ATP ratio of 20-40) and traces of ADP were also found. Comparison of the pattern of distribution of macromolecules (proteins and DNA) with the distribution of phosphocreatine between the cells and the fluid of the seminal vesicle indicates that cell lysis did not account for the phosphocreatine in the seminal vesicle fluid. Rather, the available evidence strongly suggests that this high-energy compound is actively secreted. We found that in the testes, the sperm are exposed to the highest known creatine concentration in any mammalian tissue studied. Based on these results and other recent reports, we propose that the extracellular phosphocreatine, ATP, and creatine are involved in sperm metabolism.Phosphocreatine (PCr), a guanidinophosphate, was first discovered in skeletal muscle (1,2). It is believed to serve as an energy reserve by virtue of its ability to phosphorylate ADP, leading to the production of ATP and creatine (Cr). This reversible phosphoryl transfer, mediated by creatine kinase (CK; ATP:creatine N-phosphotransferase, EC 2.7.3.2), can be represented as follows. CK/PCr-mediated energy modulator systems have since been demonstrated in several other cell types, including brain (3, 4), smooth muscle (5-7), mammalian preimplantation embryo (8), and spermatozoa (9, 10), and in the mitotic spindle of proliferating animal cells (11). In addition to an ATP-buffering role, reaction 1 has been suggested to act as an intracellular energy-transport system (12). Strong experimental evidence for such an energy-channeling role for PCr in sea urchin sperm has been produced (10). PCr and Cr in higher organisms are considered to be dead-end metabolites in that the CK-mediated reversible transfer of phosphate groups is the only known enzyme reaction in which PCr and Cr participate. However, a small portion of the total body creatine (PCr + Cr) is converted by a nonenzymatic reaction to the anhydride, creatinine, which can readily cross cell membranes and is the excretory product of PCr and Cr (13).It has been shown (14) MATERIALS AND METHODS Seminal Vesicles and Vesicular Fluid. Seminal vesicles were obtained from Swiss Webster mice (8 weeks old) or from Sprague-Dawley rats (10-12 weeks old). The animals were acclimated to a standard laboratory chow and to a 12-hr light/dark cycle for 5-7 days. They were killed by inhalation of CO2 in a large desiccator. T...