There is no specific assay for rat plasma esterase-1 (ES-1) activity. Plasma contains many esterases, while known substrates do not discriminate between esterases. With gel electrophoresis, plasma esterase isozymes can be separated. Thus, a method consisting of gradient polyacrylamide gel electrophoresis, visualization of the enzyme with a staining technique based on substrate conversion, and densitometric scanning of the stained gel has been developed for quantitative measurement of rat plasma ES-1 activity. ES-1 activities were expressed as total peak areas. Reproducibility of the method was found to be about 10% (expressed as apparent between-gel coefficient of variation). When the ES-1 zone areas was expressed relative to that of a plasma ES-1 standard, reproducibility was about 3%. The kinetics of catalysis of alpha-naphthyl acetate hydrolysis by ES-1 could be determined with the gel scanning assay; the Km was 0.76 mM. At the alpha-naphthyl acetate concentration of 2.69 mM, total peak areas of the ES-1 zone were linearly associated with the staining time (up to at least 40 min) and amount of plasma (up to 26.25 microL). The pH of the staining buffer influences the ES-1 zone area, the largest areas being obtained when the pH ranged between 7.0 and 7.8. With propionate as acyl moiety of the alpha-naphthyl ester substrate, ES-1 zone areas were higher than with either acetate, butyrate or hexanoate.