The post-nuclear fraction of rat heart tissue was fractionated by isopycnic zonal centrifugation in sucrose gradients, followed by differential centrifugation of the zonal fractions (rho-S fractionation). The distribution of 5 lysosomal acid hydrolases, a protease with neutral and alkaline activity and several marker enzymes for cell organelles (catalase, Ca2+-ATPase, cytochrome oxidase, glucose-6-phosphatase and muramidase) were studied. Three major lysosomal populations were described with equilibrium densities of 1.09, 1.17, and 1.23 gms cc-1 (omega2t = 1.54 X 10(11) rad2 sec-1), and a continuum in the size of these particles at the three different densities.