The transport of chloramphenicol (CAP) and thiamphenicol (TAP) was examined in several mammalian cell systems. Chloramphenicol was concentrated by all cells by a factor of 2 to 4 (cellular/extracellular concentration [CE] ratio). Both the uptake and efflux were rapid and temperature independent. In contrast, the influx and efflux of TAP progressed slowly at 37 degrees C over a period exceeding 30 min, the maximal C/E ratios in most instances being slightly greater than 1. Thiamphenicol was virtually excluded from cells at 0 degrees C. Cells preloaded with TAP at 37 degrees C lost little or no drug at 0 degrees C. The uptake of labeled CAP and TAP was uninfluenced by metabolic inhibitors, and was only slightly reduced by the presence of relatively large concentrations of unlabeled drug. This transport pattern is most consistent with a mechanism of simple diffusion based on partitioning of CAP in cellular compartments. It is postulated that the greater polarity of the methylsulfonyl moiety of TAP renders this drug less soluble in membrane lipids and hence more slowly diffusible. The possible significance of these findings in relation to hematologic toxicity from CAP is discussed.