High and low molecular weight (LMW) tropomyosin isoforms, by regulation of actin filaments, have a major role in the regulation of cell behaviour. They affect malignant transformation, motility, differentiation, metastasis and cell membrane protein presentation. Expression of LMW isoforms from the TPM1 and TPM3 genes have an important role in these effects but the regulation of their expression is unknown. Luciferase assays on a progressively truncated 1.7 kb fragment upstream of the exon 1b translation start site in the TPM1 and TPM3 genes in HEK-293 cells showed upstream activation sequences in TPM1 between −152 and −139 bp and in TPM3 between −154 and −102 bp. The effect of mutating candidate transcription factor binding sites identified an AML1-like transcription factor binding site in TPM1 and a cAMP response element in TPM3. Downstream from the primary activation sequence in TPM1 was a repressor region corresponding to two Sp/KLF family binding GC boxes. Band shift assays confirmed that deletion of these sites altered transcription factor binding and ChIP assays confirmed the presence of AML1 and CREB at the TPM1 and TPM3 activation sequences in the respective promoters. Expression of LMW isoforms from TPM1 and TPM3 genes is regulated very differently. This facilitates regulation of the many cell processes involving these proteins. In situations where these proteins have a critical role, such as cancer metastasis, it also facilitates specific intervention. J. Cell. Biochem. 113: 3576–3586, 2012. © 2012 Wiley Periodicals, Inc.