The distribution of mannose 6-phosphate (Man-6-P) receptors for lysosomal enzymes was investigated in Golgi subfractions prepared from three different cultured cell lines. Total microsomal fractions from clone 9 hepatocytes, normal rat kidney, or Chinese hamster ovary cells were subfractionated by flotation in sucrose density gradients, which resolves Golgi membranes into heavy (cis), intermediate, and light (trans) subfractions. The distribution of Man-6-P receptors within the subfractions was assessed by quantitative immunoprecipitation, and the results were compared to those obtained by immunoperoxidase localization of the receptors in Golgi cisternae of intact cells. In all cases, the results obtained by Golgi subfractionation and by immunoelectron microscopy were in agreement. In clone 9 cells, Man-6-P receptors were enriched in heavy (cis) Golgi subfractions, whose peak density (rho = 1.17) was greater than those containing either galactosyltransferase activity, a trans Golgi marker, or alpha-mannosidase II, a middle Golgi marker. By immunoelectron microscopy, the receptors were localized to a single cis Golgi cisterna. In Chinese hamster ovary cells, Man-6-P receptors were concentrated in Golgi membranes of low density (1.12 g/ml) overlapping the peak of galactosyltransferase activity. By the immunoperoxidase technique, the receptors were usually localized to a single trans Golgi cisterna. In normal rat kidney cells, Man-6-P receptors were found to be broadly distributed across Golgi membranes (rho = 1.12-1.17), and by immunoperoxidase localization they were found to be broadly distributed across the stacked Golgi cisternae. It is concluded that the distribution of Man-6-P receptors within the Golgi complex varies from one cell type to another. These differences in receptor distribution may reflect variations in lysosomal enzyme trafficking among different cell types.