Type III collagen is found in fetal skin and blood vessels. Previously, we characterized the proximal promoter of the human alpha1(III) collagen gene (COL3A1) using the human rhabdomyosarcoma cell line, A204, and NIH3T3 cells (Yoshino et al., Biochim Biophys Acta, 2005). In the present study, we further analyzed this promoter using additional cell lines, namely a human embryonal rhabdomyosarcoma cell line (RD) and bovine vascular smooth muscle cells (vSMCs), both of which show high expression of type III collagen. Using a luciferase assay, electrophoretic mobility shift assays (EMSA), and DNase footprinting assay, 2 types of multifactor complexes were shown to bind to the DNA region in the vicinity of the B element (- 80 to - 58), depending on the cell type. Next, we used cells stably transfected with a GFP-linked type III collagen promoter fragment for analysis of promoter expression. Usually, transfected cells retained the characteristics of the original cells. However, in several clones derived from RD cells, promoter expression as well as cell shape changed to patterns characteristic of the A204 cell line. Nuclear factors expressed by these clones were also characteristic of the A204 line.