Because of its production by adipocytes and its ability to increase preadipocyte proliferation, lysophosphatidic acid (LPA) could participate in the paracrine control of adipose tissue development. The aim of the present study was to determine which enzyme activities are involved in exogenous LPA hydrolysis by preadipocytes and adipocytes. Using a quantitative method, we observed that extracellular LPA rapidly disappeared from the culture medium of 3T3F442A preadipocytes. This disappearance was strongly slowed down in the presence of the phosphatase inhibitors, sodium vanadate and sodium pervanadate. By using [(33)P]LPA on intact 3T3F442A preadipocytes, we found that 90% of LPA hydrolysis resulted from LPA phosphatase activity biochemically related to previously described ectolipid phosphate phosphohydrolases (LPPs). Quantitative real time reverse transcriptase-PCR revealed that 3T3F442A preadipocytes expressed mRNAs of three known Lpp gene subtypes (1, 2, and 3), with a predominant expression of Lpp1 and Lpp3. Differentiation of 3T3F442A preadipocytes into adipocytes led to an 80% reduction in ecto-LPA phosphatase activity, with a concomitant down-regulation in Lpp1, Lpp2, and Lpp3 mRNA expression. Despite this regulation, treatment of 3T3F442A adipocytes with sodium vanadate increased LPA production in the culture medium, suggesting the involvement of ecto-LPA phosphatase activity in the control of extracellular production of LPA by adipocytes. In conclusion, these data demonstrate that hydrolysis of extracellular LPA by preadipocytes and adipocytes mainly results from a dephosphorylation activity. This activity (i) occurs at the extracellular face of cell membrane, (ii) exhibits biochemical characteristics similar to those of the LPP, (iii) is negatively regulated during adipocyte differentiation, and (iv) plays an important role in the control of extracellular LPA production by adipocytes. Ecto-LPA phosphatase activity represents a potential target to control adipose tissue development.