Quantitative whole-body autoradiography (QWBA) was evaluated and compared to tissue dissection/liquid scintillation counting (TD/LSC) techniques by determining the tissue distribution of radiocarbon in rats following iv administration of the antibiotic [14C]daptomycin (LY146032). QWBA, using computer-assisted video-image analysis, was initially evaluated by characterizing and calibrating commercial standards to blood and brain, kidney, liver, and lung homogenates. Frozen (carboxymethyl)cellulose blocks containing tissue homogenates spiked with [14C]glucose (370-37,000 Bq/g or 10-1000 nCi/g) were sectioned and optical densities (OD) measured. Characterization of QWBA included repeated measures data analysis to determine the significance of tissue type and intra- and inter-section and block variability. Regression models relating OD to radiocarbon concentration were also used to calibrate commercial standards for use in QWBA analyses. Results indicated that there were no substantial differences between OD readings from different tissues; however, the greatest source of variation in OD reading was section thickness. Because quantitative variations were largely attributed to section thickness, an internal standard (IS), consisting of liver homogenates spiked with [14C]glucose, was evaluated as a correction factor. Tissue concentrations of radiocarbon in male Fischer 344 rats were evaluated by QWBA and TD/LSC techniques 0.25 h following a single iv 10 mg/kg dose of [14C]daptomycin. Results indicated that tissue concentrations of radiocarbon obtained by QWBA, normalized using an IS, were comparable to those obtained by TD/LSC.