Investigation of phosphate metabolism with the aid of labeled phosphorus has been carried out, using isolated heart preparations in which only the left ventricles perform external work. The corresponding right ventricles in such preparations perform no external work with the unique experimental advantage of a single heart, providing simultaneously both working and “resting” (nonworking) metabolic states.Comparative incorporation of labeled phosphorus into nucleotide phosphate groups, non-nucleotide soluble phosphorylated components, phospholipids and ribose nucleic acid was examined as a function of cardiac work. Incorporation of the label into the rapidly exchanged high-energy phosphate groups was found to be restricted by a prior rate—limiting entry of the externally perfused isotope into the cells, thereby precluding a measure of turnover of these groups by this technic. Differences could not be demonstrated in isotope incorporation in either the phospholipids or RNA of working as compared to nonworking myocardium. It was concluded from these results that RNA is clearly not involved in energy reactions associated with cardiac work function. Phospholipid participation, on the other hand, could be clearly excluded only insofar as the phosphate moiety of these compounds is concerned.Information relevant to a mechanism of phosphate entry into cardiac cells was obtained in which a phosphorylation of hexose appeared to be involved. The composition of cardiac tissue with respect to nucleotide and non-nucleotide soluble components was examined in working and nonworkiug myocardium. Differences in composition were not found with respect to these constituents. A major component appearing in extracts of cardiac tissue was found and subsequently identified to be α-glycerophosphate. On the basis of isotope incorporation data, it was concluded that its metabolic source was of carbohydrate rather than phospholipid origin.