Changes in cytoplasmic free calcium levels ([Ca2+]i) in platelets in contact with polystyrene (PSt) and poly(acrylamide-co-methacrylic acid) (PAAmMAc) particles were evaluated and results were compared with those from two representative biological calcium agonists; thrombin and calcium ionophore A23187. PSt particles stimulated a steep increase in cytoplasmic calcium levels in platelets as much as thrombin and A23187. Serratia protease-treated platelets showed a steep increase in [Ca2+]i by PSt particles, suggesting that PSt surfaces can initiate platelet activation independent of a glycoprotein Ib (GPIb)-mediated pathway. By contrast, dibucaine-treated platelets showed little increase in [Ca2+]i by PSt particles, indicating that microfilament assembly, including binding of GPIb with actin binding protein, should be required for platelet activation in contact with PSt surfaces. PAAmMAc particles induced little increase in cytoplasmic calcium levels in platelets. However, PAAmMAc particle-treated platelets demonstrated little response to thrombin in terms of an increase in [Ca2+]i and ATP release, suggesting the possibility that PAAmMAc surfaces may regulate [Ca2+]i by influencing platelet metabolism. Furthermore, sodium azide-treated platelets showed an increase in [Ca2+]i in platelets when contacting PAAmMAc particles, supporting the suggestion that PAAmMAc surfaces could regulate platelet functions. Fluorescence polarization measurements using 1,6-diphenyl-1,3,5-hexatriene-loaded platelets revealed that PAAmMAc particles increased membrane fluidity in platelets, which may be due to physicochemical interaction with PAAmMAc surfaces.