In this study, we analyzed the effects of synthetic vitreous fibers (SVFs) on a mesothelial (MeT5A) and a fibroblast cell line (NIH3T3), compared to those exerted by crocidolite asbestos fibers. SVFs (glass wool, rock wools) do not induce significant changes in cell mortality, whereas crocidolite asbestos fibers caused a dose-dependent cytotoxicity. We investigated the correlation between the fiber-induced cytotoxicity and the extent and type of interaction of the fibers with the cell surface, and we observed that SVFs, unlike crocidolite asbestos fibers, establish few and weak interactions. Moreover, after internalization, crocidolite asbestos fibers are often found free in the cytoplasm, whereas glass wool fibers are mainly localized inside cytoplasmic vacuoles. After treatments, we also detected signs of oxidative stress, revealed by an increased reactive oxygen species (ROS) production and by an induction of superoxide dismutase (SOD) activity. The lipoperoxidative damage was characterized by a decrease in polyunsaturated fatty acids (PUFA), an increase in the content of thiobarbituric reactive species (TBARS) and a consumption of vitamin E, as a lipophilic antioxidant. Furthermore, we investigated the effect of fiber exposure on cell proliferation. and it was found that, unlike crocidolite asbestos fibers, SVFs did not induce a significant increase in DNA synthesis.