Cationic antimicrobial peptides play important roles in host defense, linking innate and adaptive immunity. hCAP18, the only human antimicrobial cathelicidin, consists of a conserved N-terminal cathelin-like domain and a C-terminal peptide, LL-37. Expression is regulated during myeloid differentiation, and tightly controlled during infection and inflammation, suggesting active regulation. Using 5' RACE (rapid amplification of cDNA ends), multiple transcription initiation sites were identified, as well as new splice variants leading to novel augmentations of hCAP18 amino acid composition in bone marrow but not peripheral blood neutrophils. Having expressed hCAP18 promoter constructs in cell lines, we found that full-length (-1739) and truncated (-978) promoter constructs had lower luciferase activities than 5'UTR deletion constructs. Transient transfection of progressively deleted constructs in the non-permissive K562 cell line led us to identify a negative regulatory element within the 53 bp immediately upstream of the ATG of hCAP18. Additionally, transient transfection of 5' deletion constructs identified a positive regulatory element within the 101 bases 5' of promoter sequence containing two GT-boxes. Negative and positive regulatory elements within the hCAP18 gene promoter provide new insights into the possible molecular basis of myeloid gene expression.