Vasoactive intestinal peptide and its G-protein-coupled receptors, VPAC-1 and VPAC-2, are highly expressed in the immune system and modulate diverse T cell functions. The human VPAC-1 5'-flanking region (1.4 kb) contains four high affinity Ikaros (IK) consensus sequences. Ikaros native protein from T cell nuclear extracts and IK-1 and IK-2 recombinant proteins recognized an IK high affinity binding motif in the VPAC-1 promoter in electrophoretic mobility shift assays by a sequence-specific mechanism, and anti-IK antibodies supershifted this complex. Stable NIH-3T3 clones overexpressing IK-1 or IK-2 isoforms were generated to investigate Ikaros regulation of endogenous VPAC-1 expression as assessed by quantifying VPAC-1 mRNA and protein. By traditional and fluorometric-based kinetic reverse transcription-PCR and (125)I-labeled vasoactive intestinal peptide binding, both IK-1 and IK-2 suppressed endogenous VPAC-1 expression in NIH-3T3 clones by a range of 50-93%. When a series of nested deletions of the VPAC-1 luciferase reporter construct were transiently transfected into IK-2 clones there was up to a 41% decrease in transcriptional activity compared with vector control. Two major IK-2 binding domains also were identified at -1076 to -623 bp and at -222 to -35 bp, respectively. As both Ikaros and its novel target VPAC-1 are highly expressed in T cells, this system may be a dominant determinant of the VPAC-1 expression in immune responses.