Nicotinic acid adenine dinucleotide phosphate (NAADP) is an endogenous regulator of intracellular Ca2+ levels in a variety of eukaryotic cell types. Although, NAADP appears to be involved in direct Ca2+ release from the ER in different cells, evidence suggests that NAADP is an atypical messenger activating a novel two-pore channels (TPCs) channel located to the endo-lysosomal cellular compartment. Evidence suggests about NAADP-mediated Ca2+ release in liver microsomes and lysosomes (Mándi et al., 2006; Zhang et al., 2007). We assume that permeabilized hepatocytes as a good model representing a tool of different Ca2+-stores (i.e. ER, mitochondria, Golgi apparatus, nucleus and lysosomes), could provide a more complex analysis of NAADP-mediated Ca2+ signaling events in these cells. Experiments were carried out on isolated rat hepatocytes permeabilized with saponine (0.1 mg/ml). To estimate the changes of internal Ca2+ concentrations, hepatocytes were loaded with 100 μM chlorotetracycline (CTC) in the dark at room temperature for 20 min. We showed that NAADP (7 μM) caused a significant decrease in CTC fluorescence intensity in permeabilized rat hepatocytes, which indicates about Ca2+ release from intracellular Ca2+ stores (31.04 ± 7.19 %; p<0.01, n=12). The simultaneous application of NAADP (7 μM) and thapsigargin (1 μM) to the incubation medium decreased the CTC fluorescence to a slightly greater extent (35.22 ± 10.64 %; p<0.05, n=6) compare to separate effects of NAADP and thapsigargin. Interestingly, NAADP did not affect fluorescence intensity after pre-incubation of hepatocytes with thapsigargin. This suggests that NAADP-mediated Ca2+ release in rat hepatocytes is thapsigargin-sensitive. Interestingly, a prior treatment of hepatocytes with thapsigargin followed by the application of nigericin led to a decrease in CTC fluorescence (29.34 ± 5.35%; p< 0.01; n = 6). Our observations revealed that in rat permabilized hepatocytes the effect of thapsigargin on ER Ca2+ level is dependent on nigericin and mediated via Ca2+ release mechanism from acidic pools. In addition, application of NAADP in the presence of the mitochondrial Ca2+ uniporter inhibitor, Ruthenium red (10 μM), did not cause statistically significant changes of fluorescence intensity. Our findings suggest that NAADP-mediated Ca2+ release from acidic pools in permeabilized rat hepatocytes is sensitive to thapsigargin and Ruthenium red.