Objective To investigate the changes of cellular configuration and polarized characteristic of visual pigment during the development of rod cells of neonatal calf in vitro. Methods Retinal cells of neonatal calf were dissociated and cultivated for 10, 20, 30 and 40 days in vitro were used for immunocytochemical analysis. Retinal rod cells were identified by rho4D2 antibody. The configuration of positive cells and rhodopsin molecular distribution were analysed at different cultivated time. The immuno-reactivity of positive cells was measured by image analysis system. Results The rho4D2 immuno-reactive cells included small round cells without protuberance and the cells with protuberance at the peak. At the 10th day after cultivation, the visual pigment immunoreaction was suffusive in the whole cell membrane and apical process; while at the 30th and 40th day, it gathered at the membrane of apical process or one pole of the cells. The results of quantitative analysis showed that the immunoreactive intensity of positive cells at the 20th day after cultivation was ber than that at the 10th day; while there was no significant difference among the immunoreactive intensity at the 20th, 30th, and 40th day. Conclusions Rod cells at the 30th and 40th day after cultivation have the polarized characterization and visual pigment molecular distribution with high level of expressive ability of protein, which are mature neurons. (Chin J Ocul Fundus Dis, 2005, 21: 394-396)