Previous studies have shown that three distinct genes encode six isoforms of tropomyosin (TM) in rat fibroblasts: the a gene encodes TM-2, TM-3, TM-5a, and TM-5b, the beta gene encodes TM-1, and the TM-4 gene encodes TM-4. Here we report the characterization of a cDNA clone encoding the most recent rat fibroblast TM to be identified, herein referred to as TM-5, that is the product of a fourth gene that is homologous to the human hTMnm gene, herein referred to as the rat slow-twitch alpha TM gene. The cDNA clone is approximately 1.7 kb and encodes a protein of 248 amino acids. Using two-dimensional gel electrophoresis, the TM-5 protein was found to co-migrate with fibroblast TM-5a and 5b. Comparison of the amino acid sequences of TM-5 to other fibroblast isoforms encoded by the alpha, beta, and TM-4 genes revealed a high degree of homology, although there were regions of divergence among the different isoforms. The gene encoding TM-5 is expressed in all tissues examined including skeletal muscle, stomach, heart, liver, kidney, uterus, spleen, brain, and diaphragm. However, Northern blot and RNase protection analyses revealed the presence of different mRNAs in fibroblasts, striated muscle (skeletal and diaphragm), and brain, which are expressed via alternative RNA splicing and the use of alternative promoters. The TM-5 protein was expressed in a bacterial system and tested for its ability to bind actin in vitro and in vivo. The apparent TM association constant (K-a) was taken as the free concentration at half saturation and was found to be 3 mu M for TM-5 compared to 2 mu M for TM-5b at an F-actin concentration of 42 mu M. When fluorescently-labeled TM-5 was microinjected into living rat fibroblasts, it localized to the stress fibers and ruffles of the leading lamella. The coiled-coil interactions of TM-5 with other low and high molecular weight TM isoforms were studied. TM-5 and TM-4 were capable of dimerizing with each other as well as with other low molecular weight isoforms (TM-5a and TM-5b), but not with the HMW isoforms (TM-1, TM-2, and TM-3). In addition, TM-5a and TM-5b were unable to heterodimerize with each other. The implications of these results in understanding the role of TM diversity in cytoskeletal dynamics are discussed. (C) 1996 Wiley-Liss, Inc.