Mitochondria1 response to the effect of a hydroxylase (PH) inhibitor was tested in marrow stromal cells during stimulation of osteoprogenitor cell (OPC) differentiation. The rationale for this was to explore pathways of regulatory interactions between procollagen synthesis and mitochondrial respiration that could be linked to the commitment of OPCs to mineralization. Stimulated OPCs exposed to the PH inhibitor cis-hydroxyproline (cis-HP), compared to the noninhibiting isomer trans-HP, for 11 days showed a dose-dependent decrease in cell proliferation, the surviving cells showed increased alkaline phosphatase activity. Trans-HP did not influence the cis-HP effect on ALP and on proliferation arrest. Short time exposures, 2-3 days, to cis-HP at different periods suggested that Days 0-3 and 3-5 were critical for the commitment to Day 21 mineralization of OPCs. On Days 0-3 cells were most sensitive to cis-HP, since on Day 11, 8 days after removal of cis-HP, they were too scarce to be counted by the staining method. However, the presence of 5.0 mM cis-HP in the cultures during Days 3-5 has induced on Day 21 close to 24-fold more mineralization/cell than controls, compared to the trans-HP effect, which was only close to %fold. The presence of cis-HP in the cultures on Days 0-3 has augmented the mitochondrial Day 3 retention of rhodamine 123 (Rho) in the stromal cells, relative to controls, compared to the presence of trans-HP. However, the presence of cis-HP during Days 3-5 or 3-6 resulted in lower Day 5 Rho retention, relative to controls, which was not significantly different from the retention that resulted from trans-HP. Since Rho retention is related to the final result of aerobic respiration level, these results are interpreted as a cis-HP inhibitory effect on procollagen peptidyl-proline hydroxylation, which may in turn release oxygen surpluses, to be available for mitochondrial consumption. The fall in Rho retention responses to cis-HP between Days 0-3 and 3-5 is suggesting either abrupt decrease in proline hydroxylation or poor mitochondrial sensitivity to oxygen in Day 3-5 cultures.