Secondary lysosomes were isolated from rat liver and separated into a soluble and a membrane fraction. Plasma membranes and microsomes were also isolated and antisera against the various fractions were prepared in rabbits. Lysosomal content and detergent-solubilized membrane fractions were analysed in two-dimensional immunoelectrophoresis (crossed immunoelectrophoresis). The immunoprecipitates were stained by histochemical procedures for different enzyme activities such as phosphatases, non-specific esterase, arylsulphatase, glycosidases and L-leucyl-beta-naphthylamidase. When lysosomal content was tested against its corresponding antiserum, 17 different precipitates could be seen. Most of the enzyme activities tested were shown to reside separately in one or a few precipitates each. In contrast, when the membrane extracts were investigated, a more polymorphic pattern of enzyme-active precipitates appeared. Thus, when lysosomal membrane extracts were reacted with homologous antiserum 11 precipitates with acid phosphatase activity were obtained. Several of the antigens were electrophoretically different and immunologically non-identical. As expected from the biology of secondary lysosomes, many of their antigens were also found in microsomes and/or plasma membranes, but several antigens unique for lysosomes were detected concomitantly. Closer analysis of these results indicated that several seemingly identical enzyme-active proteins occurred both in soluble and membrane-associated forms. However, while many of the membrane antigens expressed 2-4 different enzyme activities, only one activity was detected in individual precipitates of the lysosomal content. Thus, acid phosphatase activity was found together with esterase activity in three membrane-associated antigens. The precipitates formed by two of these also stained for arylsulphatase and nucleoside tri-, di- and monophosphatase activities. L-Leucyl-beta-naphthylamidase activity was found in one additional acid-phosphatase-active precipitate.