An approach is presented for determining relative rates of 3H-thymidine incorporation in S-phase for discrete ploidy subpopulations that coexist in heteroploid tumors. The analysis is facilitated by an automated imaging methodology developed with a Quantimet 720 System for simultaneous determination of Feulgen-stained DNA content and grain counts of 3H-thymidine-labeled cells in Feulgen-stained autoradiographs (Sklarew RJ: J Histochem Cytochem 30:35, 30:49, 1982). The experimental design involves 3H- and 14C-thymidine double labeling in the presence of Colcemid. Data from heteroploid rat sarcoma cultures illustrate the profound influence of differential 3H absorption and relative emulsion efficiency. These factors are controlled, respectively, through measurements of mean nuclear optical density and grain density. This is essential for a valid comparison of grain counts between cells that differ in their autoradiographic geometry. Thus, relative DNA specific activity may be assessed in individual cells. The protocol and analysis supports a simultaneous evaluation of the S-phase ploidy composition and S-emptying profile, as presented in a companion article (Sklarew RJ: J Histochem Cytochem 32:413, 1984).