Cell culture studies have shown that NSAID may influence osteogenic activities of osteoblast cultures. However, these studies did not consider long-term effects on differentiating cells. The influence of Voltaren with the non-steroidal agent diclofenac on proliferation and gene expression of the osteoblast-like cell line SaOS-2 was investigated 2, 9, and 16 days after incubation. Two days after 24 h of incubation, 50 microg/ml diclofenac reduced the proliferation and collagen type I expression while 9 and 16 days later no effect was found on either of the parameters. In contrast, 50 microg/ml NSAID has no effect on alkaline phosphatase expression 2 days after incubation while 9 and 16 days later expression had been reduced. Lower concentrations (1.56 and 0.19 microg/ml) had no effects on the studied parameters. BrdU and MTT test showed that 50 microg/ml diclofenac reduced proliferative and metabolic activity. Lower concentrations (< or =25 microg/ml) had a lower or no influence. The findings indicate that the NSAID impairment depends on cellular differentiation stage and is not confined to the time during or immediately after NSAID incubation. According to these results in vitro testing of drugs should be performed over a longer time period to detect possible long-term impacts.