Tartrate‐resistant acid phosphatase (TRAP) is an iron‐binding protein that is highly expressed in osteoclasts. To characterize the regulation of TRAP gene expression, progressive 5′ and 3′ deletions of a 1.8 kb fragment containing the 5′‐flanking sequence were fused to a luciferase reporter gene. Two nonoverlapping regions of this 1.8 kb fragment had promoter activity. The upstream promoter (P₁) was located within the region from –881 bp to –463 bp relative to the ATG, while the downstream promoter (P₂) was located between –363 bp to –1 bp in a region we have previously shown to be an intron in transcripts originating from the upstream promoter. A putative repressor region for the P₂promoter at –1846 bp to –1240 bp and a putative enhancer region at –962 bp to –881 bp relative to the ATG were identified. PCR analysis of promoter‐specific transcription of the TRAP gene in various murine tissues showed that both promoters were active in several tissues. Transferrin‐bound iron increased P₁promoter activity 2.5‐fold and hemin decreased P₁promoter activity, but neither had any effect on P₂ activity. These data show that the transcriptional regulation of the TRAP gene is complex and that iron may play a key role in TRAP gene regulation.