A method for isolation of rat hepatocytes using liver perfusion by ethylenediamine tetra‐acetic acid (EDTA)‐containing sucrose solution and mechanical tissue disaggregation by controlled vibration (MVD) is described. The yields of hepatocytes produced by this method were similar to those obtained using collagenase perfusion. The cells had well preserved membrane integrity as judged by the trypan blue staining test (91±4%), ATP contents, rates of endogenous respiration and enzyme leakage that indicated they were functional cells. There was little evidence of expression of latent damage when the cells were stored either at 37°C (by pre‐incubation) or at 4°C. This method can be used to isolate high yields of functional cells from rat liver if the collagenase perfusion technique is not available.