The enamel organ of growing rat incisors was perfusion-fixed with a mixture of formaldehyde and glutaraldehyde and processed for ultracytochemical demonstration of ouabain-resistant, K+-stimulated p-nitrophenylphosphatase representing the second dephosphorylative step of H-K-ATPase by use of the one-step lead method. Throughout the stages of amelogenesis, the enzymatic activity was found in the plasma membranes, mitochondrial membranes, and lysosomal structures of the cells of stratum intermedium, papillary layer, and ameloblast layer. Gap junctions and desmosomes between these cells were, however, free of reaction product or showed slight precipitates of reaction. The stellate reticulum and the outer enamel epithelium at the stage of enamel secretion were usually negative for reaction. Although secretory, transition, and ruffle-ended maturation ameloblasts showed enzymatic activity at their basolateral cell surfaces, their distal cell surfaces facing the enamel were always free of reaction product. On the other hand, the smooth-ended maturation ameloblasts seldom showed a positive reaction, except in lysosomes and along their basal cell surfaces. An energy-dispersive X-ray microanalysis of reaction products of H-K-ATPase in unosmicated tissue sections demonstrated that they were composed of lead and phosphorus, which had been released during the dephosphorylation of substrate. In cytochemical controls, the enzymatic activity was completely dependent on substrate and potassium ion, resistant to ouabain and levamisole, and inhibited by nolinium bromide, a specific inhibitor of H-K-ATPase. In addition, inorganic trimetaphosphatase as enzymatic marker of lysosome was localized in dark and pale lysosomes, phagosomes, multivesicular bodies, and ferritin-containing vesicles of the ameloblasts and the cells of stratum intermedium and papillary layer. These membrane-bound structures were also positive for H-K-ATPase reaction. These results suggest that: 1) H-K-ATPase functions to maintain an acidic internal pH of lysosomes in the enamel organ cells; and 2) H-K-ATPase localization in the plasma membranes of enamel organ cells is concerned with efflux of protons derived from cytoplasmic water.