To delineate the temporal and spatial acquisition of the smooth muscle of the ureter, Sprague-Dawley rat embryos and newborn pups were immunostained with alpha-smooth muscle actin (alpha-SM actin) antibody. alpha-SM actin expression was first detected in the urinary tract at 16 days of gestation (E16) in a thin subserosal zone about the urogenital sinus. At this time, the E16 ureter is composed of a simple cuboidal epithelium which is surrounded by 1 to 2 layers of condensed alpha-SM actin negative spindle cells. No immunostaining was detected along the ureter or its intrarenal branches until the 20th day of gestation (E20). alpha-SM actin expression in the E20 ureter exhibited regional differences. The number of alpha-SM actin positive smooth muscle cells was greatest in the distal ureter, intermediate in the mid ureter, and least in the proximal ureter near the kidney. While smooth muscle formation in the bladder was subserosal, in the ureter it was subepithelial. During postnatal life, alpha-SM actin expression increased in both organs as all periepithelial spindle cells stained positive and intensified their staining. Smooth muscle differentiation of the ureter and bladder occurs later in embryonic life than other visceral and vascular organs and occurs in an ascending fashion from the bladder to the intrarenal collecting system. It is likely that the activation of visceral smooth muscle myogenesis within the urinary tract is governed by positional information specific to the embryonic development of each organ.