The expression of aldolase A L-type mRNA is increased in growth-arrested mouse NIH3T3 cells and remarkably down-regulated in actively proliferating cells. Treatment of proliferating cells with cycloheximide abolished the down-regulation of L-type mRNA expression, thus indicating that a protein factor acts as repressor in proliferating cells. Transient transfection experiments in NIH3T3 cells showed that a negative regulatory cis-element (NRE) is involved in the modulation of the transcriptional activity of the distal L promoter. The repressor, which is a protein of approximately 97 kDa, binds the murine aldolase A NRE, revealing a much more intense DNA-protein complex in proliferating NIH3T3 cells than in serum-deprived cells. Mutations in the negative regulatory cis-element showed that the GA-rich motif is required for protein binding and silencer function. We conclude that the expression of L-type mRNA is modulated by the interaction between a cell cycle-dependent DNA-binding protein and the murine aldolase A NRE.