Cells from the muscular layer of neonatal (3-day-old) rabbit urinary bladders were dissociated with collagenase, and cultured in M199 supplemented with 10% fetal bovine serum and antibiotic-antimycotic. Cells in culture were of two types: long and short. The short cells were thick and spindle-shaped, and the long cells were flat and elongated. The long cells can be about 15 times longer than the short cells. The short cells do not divide, but the long cells divide readily. Expressions of smooth muscle and non-muscle myosins, alpha-smooth muscle actin, vimentin, and h-caldesmon were determined by immuno-fluorescence microscopy using specific antibodies. Both types of cells react strongly with antibodies against smooth and non-muscle myosins. Unlike the short cells, the long cells also contain alpha-actin and vimentin. The expression of h-caldesmon was very weak in both cell types. Also, cells dissociated from the smooth muscle layers of adult (6-month-old) rabbit bladder were cultured under the same conditions as the cells from the neonatal bladders to see if the heterogeneity of smooth muscle cells, exhibited by cells from neonatal rabbits, is also shown by cells from adult bladder. Two types of cells were also identified. The cells were then fixed and examined with the same panel of antibodies that we used for the neonatal cells. The long cells from adult bladder muscle express similar proteins to those in the neonatal long cells, and the short cells were stained positively with smooth muscle myosin, non-muscle myosin, alpha-smooth muscle actin, and lightly with caldesmon. Although the absence of vimentin in the short cells from adults is similar to that from neonatal, the strong expression of alpha-actin in the adult short cells is unlike the short cells from neonatal rabbits, in which their expression is barely detectable.