The objective of this study was to evaluate the capability of a photothermal (PT) assay in determining the effects of graded doses of nicotine in a pancreatic acinar cell line (AR42J). The cellular response to nicotine was detected through the monitoring of PT signals from light-absorbing endogenous cellular structures that have been used as natural indicators for nicotine's action. It was demonstrated that introducing nicotine to cultured acinar cells in vitro leads to changes in cellular absorbing structures, thereby altering the microstructure of PT cell images and the temporal shape of PT signals. The results showed that the dependence of specific PT parameters was almost proportional to nicotine concentrations ranging from 1 nM to 100 microM, with the saturation maximum at and around 100 microM - 1 mM; thereafter, PT signals decreased rapidly to control levels and even lower, in the range of 1 - 50 mM. Conventional fluorescent tests (Annexin V--Propidium Iodide) performed in parallel showed no effect with nicotine at a concentration <1 microM (three orders of magnitude greater than the sensitivity threshold of the PT assay). With an increase in nicotine concentration from 1 mM to 50 mM, rapidly growing apoptotic and necrotic cells were detected. Thus, the PT assay demonstrated the capability for high-sensitivity detection of nicotine's impact, which may be related to a change in cell metabolism, apoptosis, or necrosis, depending on nicotine concentration.