Butylated hydroxyanisole (BHA) is perhaps the most extensively used synthetic antioxidant in the food and cosmetic industry, although considerable controversy exists in the literature regarding the safety of this compound. Most in vitro studies describing the effects of BHA have been performed in cancer cells, but it is unclear whether normal cells are equally susceptible to BHA exposure. The present study investigate the toxic potential of BHA in mammalian cells, using biochemical and morphological parameters, which reveal interference with structures essential for cell survival, proliferation and/or function. Cell growth inhibition was assessed by using colorimetric assays, whereas cellular alterations after BHA exposure, were evaluated using conventional light and fluorescence microscopy. Low doses of BHA exerted a significant cytotoxic effect, associated with loss of mitochondrial function. As the concentration of BHA was increased, morphological alterations in critical subcellular targets such as lysosomes, mitochondria and actin cytoskeleton, were observed. In parallel, BHA induced an irreversible loss of cell proliferative capacity, preceding apoptosis induction. Thus, the dose-dependent activity of BHA on Vero cells appears to be cytotoxic as well as cytostatic. Our observations, although simplified with respect to the in vivo situations, allowed the assessment of the specific damage at the cellular level, and provide some clue about the effects of BHA in non-tumoral mammalian cells.