The subcellular distribution of Na,K-ATPase in rat lacrimal gland acinar cells was surveyed by subcellar fractionation followed by determination of two Na,K-ATPase catalytic activities, K(+)-dependent p-nitrophenylphosphate hydrolysis and ouabain-sensitive ATP hydrolysis, by Western blotting of isolated membrane fractions, and by analysis of lacrimal tissue with immunocytochemical methods at the light and EM levels. Both catalytic activities distributed in parallel after centrifugation on sorbitol gradients. They were associated with membrane samples that appeared to be derived from: (1) acinar cell basal-lateral membranes; (2) the Golgi complex; and (3) endocytic compartments. beta 1-subunit immunoreactivity closely paralleled catalytic activity. The alpha 1-subunit immunoreactivity distribution suggested the presence of alpha 1-subunits in alpha beta complexes and excess alpha 1 subunits which were not assembled with beta subunits and not catalytically active. In the putative basal-lateral membrane and endocytic samples, alpha 1-reactivity was associated primarily with a 100-kDa band, while in the Golgi samples it was associated primarily with 40 and 60-kDa bands. beta 1-reactivity was also heterogeneous, with reactivity in basal-lateral membrane and putative endocytic samples associated with a broad band of 50-54 kDa, and reactivity in Golgi samples associated with discrete bands of 50, 52, and 54 kDa. Staining with anti-holoenzyme and anti-alpha 1-subunit antibodies yielded strong indirect immunofluorescence signals both in plasma membrane and in intracellular regions of acinar cells. beta 1-like immunoreactivity was concentrated in cytoplasmic regions of acinar cells. Immunogold electron microscopy revealed positive staining with anti-holoenzyme in Golgi membranes of acinar cells and in basal-lateral membranes of duct cells. These data support the hypothesis that lacrimal acinar cells contain substantial cytoplasmic pools of Na,K-ATPase and that there is a location-dependent heterogeneity which is not detected by immunocytochemical methods.