A kinetic assay for glycerol based on the glycerokinase and firefly luciferin-luciferase reactions was developed for the purpose of allowing automatic analysis of large series of incubates from human fat cells. The method includes simplified sampled pretreatment and fully automated analysis, recording, and calculation of data. The upper limit of the dynamic range of the assay was extended from a glycerol concentration in cuvette of 2 up to 100 microM. This is due to a principally new method to linearize the nonlinear part of the standard curve. Recovery experiments reveal 100% glycerol recovery. Within-run and between-run precisions are high with coefficients of variation of between 1.2 and 3.7%. Samples are stable for up to 6 months in an ordinary freezer. The detection limit expressed as 2 SD of the blank result is 0.07 microM glycerol in cuvette, which is slightly lower when compared to the previous luminometric methods. The apparent blank, which is typically 0.4-1.0 microM glycerol in cuvette, has shown significant contributions only from bovine serum albumin, 0.03 microM, and glycerokinase, 0.01 microM. The analytical performance and degree of automation of the glycerol method described in this paper makes it well suited for serial studies of lipolysis in human fat cells in the presence of lipolytic or antilipolytic agents.