Single C motif-1 (SCM-1)/lymphotactin is a C-type chemokine whose expression is activation dependent, cyclosporin A sensitive and restricted to CD8+ T cells, double-negative thymocytes, gammadelta-type T cells, and NK cells. In humans, there are two highly homologous genes encoding SCM-1alpha and SCM-1beta. Here we examined the regulatory mechanism of the SCM-1 genes. The luciferase reporter gene under the control of the 5' flanking region of 0.7 kb was strongly induced upon activation with anti-CD3 or PHA plus PMA only in SCM-1-producer T cell lines through a cyclosporin A-sensitive mechanism. An element termed E1 located at -108 to -95 nt relative to the major transcription start site was found to be critical for the promoter activity. In electrophoretic mobility shift assays using the E1 oligonucleotide as probe, nuclear extracts from unstimulated T and B cell lines formed a constitutive complex termed complex I, while nuclear extracts from stimulated SCM-1-producer T cell lines formed a higher mobility complex termed complex II with a concomitant decrease in complex I. The shift from complex I to complex II seen only in SCM-1-producer T cell lines upon activation was completely suppressed by cyclosporin A. Both complexes were critically dependent on the NF-AT core sequence TTTCC in the E1 element and were partially supershifted by anti-NF-ATp. One-hybrid assays in yeast isolated NF-ATp as an E1 binding protein, and transfection of NF-ATp into T and B cell lines strongly enhanced the activation-dependent SCM-1 promoter activity. Collectively, a unique mechanism involving NF-ATp appears to regulate the cell type-specific and activation-dependent expression of the SCM-1 genes.