The ability to serially propagate mammalian cells in microcarrier cultures is essential for large‐scale operation. The success of such serial propagation depends on viable dissociation of cells from microcarriers and the normal growth and product formation after subsequent reinoculation. The high pH treatment developed for dissociating cells from DEAE‐derivatized microcarriers was not as effective for a number of cell strains cultivated on gelatin‐coated microcarriers. By prewashing the cell‐laden microcarriers with buffer containing a chelating agent, bovine kidney cells, BK, human embryonic foreskin fibroblasts, FS‐4, and continuous human kidney cells, TCL‐598 which produces prourokinase, were viably dissociated from commercially available gelatin‐coated microcarriers, Cytodex‐3. Cells dissociated from microcarriers reattached and grew on micro‐carriers subsequent to inoculation into subcultures. However, after subculturing, cells may attach at different rates to newly added beads and to conditioned microcarriers which cells had previously grown. It resulted in an uneven cell distribution on microcarriers and inferior growth kinetics. This effect was more profound for BK and FS‐4 cells which are propagated with a low multiplication ratio. Specifically, BK cells attach to conditioned beads at a faster rate than to new beads, while FS‐4 cells attach to new beads faster than to conditioned beads. Thus, for these two cell strains, a separator was used to separate the microcarriers from the suspension of dissociated cells before subsequent inoculation. For TCL‐598 cells, which are propagated at a high multiplication ratio, this dissociation technique can be applied directly without the separation of dissociated cells and conditioned microcarriers. All the three cell lines tested exhibit normal growth kinetics in serial propagation on microcarriers. Furthermore, the production of prourokinase by TCL598 cells serially propagated on microcarriers was comparable to that inoculated from roller bottles.