We compared the cytoplasmic pH (pHi) of quiescent and actively cycling thymic lymphocytes. Human and rat thymocyte suspensions were fractionated by centrifugation on one-step albumin density gradients. The pellet was composed of small, quiescent cells and the interphase contained mostly larger, actively cycling cells with a high proliferation index. When measured using ['4C]-dimethyloxazolidinedione (DMO), pHi in t h e large cells of both species was approximately 0.15 units more alkaline than in the small cells. However, these differences were not detectable when pHi was measured with carboxylated fluorescein derivatives generated in situ by cytoplasmic enzymes. This apparent discrepancy can be explained by compartmentation of DMO, which accumulates in the alkaline mitochondrial matrix. Comparison of the mitochondrial content of quiescent and cycling thymocytes by several methods showed that the latter contained over 2.5-fold more mitochondria per unit cell volume. Assuming a constant intramitochondrial pH, this difference can account for the observed accumulation of DMO (i.e., apparent cytoplasmic alkalinity) in the actively proliferating cells. Therefore, no evidence was found for the existence of differences in pHi between quiescent and proliferating lymphocytes. Moreover, caution must be exercised when comparing DMO partition data in cells with varying relative mitochondrial content.