The objective of this study was to demonstrate that tissue concentrations of radioactivity derived by digital analysis of autoradiograms were comparable to values derived from direct sampling and analysis of tissues. In addition, we describe the preparation and calibration of standards for use in quantitative whole-body autoradiography. For this study, three male Long-Evans hooded rats were administered 14C radioactivity intravenously. The animals were sectioned for whole-body autoradiography, with concomitant sampling of blood and 16 selected tissues. After 3 weeks of film exposure, the optical densities of the resulting autoradiograms were analyzed with a RAS3000 digital imaging system to estimate tissue concentrations of radioactivity. These concentrations were then compared with those obtained by direct analysis of the tissue samples. The concentrations derived from digital analysis of the autoradiograms were very highly correlated with those determined from direct tissue analysis (r = 0.956). Linear regression analysis yielded a straight line with a slope of 0.97 and a goodness of fit (r2) of 0.913. This analysis suggested that there is an approximate 1:1 correlation between concentration values determined by the two methods. Marked differences between the values derived via the two techniques were observed for only three tissues. However, this subset of the data accounted for only 6% of the total data, and the differences were probably due to contamination from adjacent tissues during excision. Overall, the concentrations of radioactivity derived from digital analysis of the autoradiograms were comparable to those derived from direct analysis of tissue samples. The results indicated that the digital analysis procedure for film can serve as a valuable adjunct to conventional tissue analysis for radioactivity.