Using glycerol kinase and [3H]glycerol, a kinetic isotope dilution assay for glycerol has been developed. Reactant and product are separated by stepwise elution from QAE-Sephadex. This assay is sensitive to as little as 100 pmol of glycerol, avoids numerous drawbacks of the traditional fluorescent assay, and readily detects glycerol production by fewer than 10(5) cardiomyocytes.