It was identified that a Spl-like cis-element in the adipocyte amino acid transporter gene (AAAT) promoter is the major cis-motif for its induced expression during the 3T3-L1 preadipocyte differentiation process. Electrophoretic mobility shift analysis of this cia DNA element showed that the transcription factors binding to this sequence were Sp1 and Sp3. Protein analysis of Sp1 and Sp3 in nuclear extracts from 3T3-L1 cells at various differentiation stages indicated that these two transcription factors existed in noninduced 3T3-L1 preadipocytes, but they did not bind to the AAAT promoter element with high affinity. They were activated after differentiation induction. It was further demonstrated that dephosphorylation of Sp1 increased its binding affinity to this inducible AAAT promoter element.