We previously cloned a gene for a novel myosin (called MysPDZ) containing a PDZ-domain from bone marrow stromal cells. This new myosin is found in humans and classified as one of the class XVIII myosins (Myo18A). Here, we report the hematopoietic cell-specific splicing isoform (MysPDZbeta) in addition to the previously reported isoform (MysPDZalpha). Combined with mouse genome sequence data, the overall genome structure and generation of the two spliced isoforms are deduced. The MysPDZbeta protein lacks a PDZ-domain in the N-terminal region. Studies of the subcellular localization of the two spliced isoforms indicated that MysPDZalpha containing the PDZ domain co-localizes with the ER-Golgi complex, while MysPDZbeta, which lacks the PDZ domain, localizes diffusely in the cytoplasm. These results suggest that the isoforms differ in their subcellular localization and may have different functions in membrane ruffling and membrane traffic pathways. The PDZ-containing spliced isoform (MysPDZalpha) is not expressed in bone marrow hematopoietic cells, whereas MysPDZbeta lacking the PDZ is specifically expressed in most hematopoietic cells. It is noted that neither isoform is expressed in red blood cells. Interestingly, MysPDZalpha was detected in mature but not in immature macrophages, and its level increased after the induction of differentiation of M1 cells, suggesting a functional role of PDZ-containing myosin in macrophages.