The objectives of this study were to elucidate the influence of occlusal hypofunction on the histological structure of periodontal ligament (PDL) and on the cell proliferation and alkaline phosphatase (ALP) activities of cultured PDL cells isolated from PDL. Forty-two male Wister rats were divided into three groups: two experimental groups and one untreated control group. Occlusal hypofunction was generated in the molar region in the experimental groups for 3 or 7 days. Paraffin sections of the upper molar region were stained with H.E. or ALP for light-microscope observation. PDL cells isolated from molars of each group were cultured for 7 days for cytological analyses. Cell proliferation (BrdU labeling) and ALP activity of the three groups were compared. Change in the width of the PDL space, loss of functional arrangement of PDL fibers and reduction of PDL cell number were observed among molars of the experimental groups compared with the control group. ALP activity was increased in the molar region where significant enhancement of bone morphogenesis had not yet occurred. The ratio of BrdU positive cells was significantly lower and ALP activity was higher than that of the control group among cultured cells from molars of the experimental groups and the control group. The results clearly showed that occlusal hypofunction alters the histological characteristics of PDL even in a short time. Occlusal hypofunction reduces the cell proliferation and increases the ALP activity of PDL cells. The influence of occlusal hypofunction memory on cells is retained for at least 7 days in vitro.