The OECD guideline (TG129) recommends mouse fibroblasts (3T3) & normal human epidermal keratinocytes (NHK) for performing in vitro cytotoxicity assays but also mentions limitations of these cells, such as having little to no metabolic capacity or possibility of inappropriate interpretation of data if these cells are unrelated to the target organ of interest. Considering the primary target organ for inhalable products, we conducted this study to evaluate the suitability of a human derived alveolar cell line A549 for the neutral red uptake (NRU) assay under various testing conditions. First, positive control sodium lauryl sulfate (SLS) and DMSO extracted cigarette smoke total particulate matter (TPM) were tested for cytotoxicity in A549 and 3T3 under submerged condition. Secondly, A549 were grown at the air-liquid interface (ALI) and assessed for cytotoxicity following 24 and 48 hr. of treatment with SLS in media supplied from basal side, containing 0% or 5% serum. Dose-dependent cytotoxicity was observed in 3T3 and A549 in response to both SLS and TPM and the corresponding IC50 were estimated. Under submerged conditions containing serum, A549 displayed lower IC50 (SLS) and higher IC50 (TPM) in comparison to the values of mouse 3T3 cells, suggesting species-dependent differences in sensitivities. No significant difference in cytotoxicity was observed in A549 cells between submerged and ALI cultures in response to SLS. Enhanced cytotoxicity was evident in A549 cells exposed to SLS under serum free environmental conditions. In summary, 1) A549 cells could be effectively used in the TG129 NRU assay in submerged as well as ALI conditions and 2) media composition such as presence of serum, should be carefully controlled especially when using human-derived cells such as A549.