We have reported previously that cytosolic pH (pHi) in the mouse 2-cell conceptus is controlled by a H+-monocarboxylate cotransporter (MCT) that is sensitive to cinnamates and p-chloromercuriphenylsulfonate. In the present study we have used measurement of pHi with BCECF to characterize the changes in MCT activity during pre-implantation development. We found that the resting pHi in bicarbonate-free conditions increased significantly from the unfertilized oocyte to the 2-cell stage, but thereafter remained constant. There was no evidence for changes in MCT activity during the cell cycle, but MCT activity was found to increase during development. Using RT-PCR we demonstrated that mRNA encoding MCT isoforms 1, 2 and 3 is present throughout pre-implantation development. The inhibitor of MCT1, p-chloromercuribenzoic acid, completely abolished the effect of extracellular l-lactate on pHi suggesting that MCT1, and not MCT2, plays a functional role in pHi regulation in mouse conceptuses, while the role of MCT3 remains unclear. We further found that removal of glucose from the culture medium, which has previously been shown to stimulate pyruvate uptake by blastocysts, had no effect on the activity of the MCT. These findings suggest that the changes in pyruvate uptake that have been observed following compaction are not due to changes in the activity of the MCT. These findings indicate the presence of MCTs during early embryonic development.