The tibiae and fibulae of 14-week-old rats were subjected to a single 5 minutes period of cyclic longitudinal loading at 1 Hz. The activity of the enzymes glucose 6-phosphate dehydrogenase (G6PD) and alkaline phosphatase (ALP) in osteocytes and periosteal osteoblasts was measured immediately and 24 h after loading. In osteocytes G6PD activity was increased immediately after loading but returned to control values 24 h later. There was no detectable ALP activity in these cells regardless of loading history. In periosteal osteoblasts G6PD activity was raised immediately after loading and remained higher than controls 24 h later. ALP activity in periosteal cells was unaffected immediately after loading but 24 h later was substantially increased. These findings are consistent with osteocytes and periosteal cells both being immediately responsive to periods of intermittent loading in their adjacent matrices. In both cell types an early feature of this response is an increase in G6PD activity. In osteocytes this response is short-lived, suggesting that it is an early biochemical change associated with strain perception that does not progress to matrix synthesis. The increase in G6PD activity with unaffected ALP levels in periosteal cells immediately after loading is consistent with a similar response. In these cells the increase in G6PD accompanied by increased ALP levels 24 h after loading suggests that the loading-related response progresses to new bone formation.