Summary: The gene for the human leukocyte-specific transcript 1(LST1) encodes a small protein that modulates immune responses and cellular morphogenesis. The LST1 transcripts are expressed at high levels in dendritic cells. Because of the complex splicing pattern, use of alternative 5'-UT exons, and a biologically interesting pattern of expression of LST1 mRNA, we studied the human LST1 gene promoter and regulatory elements. We identified an additional upstream 5'-UT exon in U937 monocytic cells. Transient transfection studies demonstrated that the combination of regions from -1363 to -621 with -112 to -54, relative to the translation start codon, produced the highest level of transcripts from among the various constructs tested, but the pattern of transcripts produced was only a subset of those produced from the endogenous gene. DNase I footprinting analysis and electrophoretic mobility shift assays (EMSA) showed that oligonucleotide probes corresponding to three regions, -1171 to -1142 (BI), -1136 to - 1111 (BII), and - 783 to - 751 (BIV), bound proteins in U937 nuclear extracts. Competition and super-shift EMSA did not identify any known transcription factors responsible for BII probe binding. These studies suggest that a novel DNA binding site and interaction of multiple regulatory elements may be involved in mediating the expression of the various forms of LST1 mRNA.