We have explored the possibility of increasing the sensitivity of the mixed-phase assay for chloramphenicol acetyltransferase (CAT) by using a low concentration (3.75 microM) of isotopically undiluted [3H]acetyl-CoA (200 mCi/mmol). Using extracts of PC12 cells transiently transfected with a plasmid CMV-CAT, we found that the assay was linear with time for about 8 h, unless 25% of the substrate was exhausted. Under the conditions of the assay, the tritiated substrate was relatively stable, as 75% was still available for the reaction after a 20-h incubation at 37 degrees C under the toluene phase in the absence of cell extract. CAT activity could be reliably measured with 4-8 ng protein of cell extract, corresponding to 50-100 transfected cells. We determined the range of linearity of the initial rate with the volume of cell extract and showed that, above a certain value, the rate becomes limited by the diffusion of 3H-acetylated chloramphenicol in the organic phase. The sensitivity of the new assay compared favorably with that of the previously described CAT assays and approached that of the luciferase assay.