Nine malignant breast epithelial cell lines and 3 normal breast cell lines were examined for stress fiber formation and expression of TPM1 isoform-specific RNAs and proteins. Stress fiber formation was strong (++++) in the normal cell lines and varied among the malignant cell lines (negative to +++). Although TPM1γand TPM1δwere the dominant transcripts ofTPM1, there was no clear evidence for TPM1δprotein expression. Four novel human TPM1 gene RNA isoforms were discovered (λ,μ,ν, andξ), which were not identified in adult and fetal human cardiac tissues. TPM1λwas the most frequent isoform expressed in the malignant breast cell lines, and it was absent in normal breast epithelial cell lines. By western blotting, we were unable to distinguish between TPM1γ,λ, andνprotein expression, which were the only TPM1 gene protein isoforms potentially expressed. Some malignant cell lines demonstrated increased or decreased expression of these isoforms relative to the normal breast cell lines. Stress fiber formation did not correlate with TPM1γRNA expression but significantly and inversely correlated with TPM1δand TPM1λexpression, respectively. The exact differences in expression of these novel isoforms and their functional properties in breast epithelial cells will require further study.