Previous work from this laboratory has shown that the serotonin (5-HT) induced response is significantly augmented in differentiated NG108-15 (NG) cells treated with dibutyryl cAMP (Bt2cAMP) due to qualitative and quantitative changes in the expression of the 5-HT3 receptor as demonstrated by specific [3H] LY-278584 (a selective 5HT3 receptor antagonist) binding. In this study, we investigated whether there is any change in the relative expression of the 5-HT3A and 5-HT3B subunits in NG cells differentiated following Bt2cAMP treatment cells. The major findings of this study were that the relative amount of 5-HT3B subunit mRNA in Bt2cAMP-treated NG cells 5 days following Bt2cAMP-treatment was greater than that in the untreated cells. In contrast, the relative expression of the 5-HT3B subunit protein in the Bt2cAMP-treated NG cells was much less than in the untreated cells, but the relative expression of the 5-HT3A subunit in the Bt2cAMP-treated NG cells was similar to the untreated cells. Therefore, no relationship between mRNA and protein expression for 5-HT3A and 5-HT3B subunits in Bt2cAMP treated and untreated NG cells were observed. It was also found that fluorescent intensity for the 5-HT3B subunit in the cell body of the Bt2cAMP treated and untreated NG cells gradually decreased from the day 1–5 after Bt2cAMP treatment. However, in specific areas such as the varicosity and nerve endings of the Bt2cAMP treated cells, staining intensity for the 5-HT3B subunits was stronger than in the untreated cells at the all time points, peaking at day 5 post-treatment. These results suggest that the augmented response induced by 5-HT acting via 5-HT3 receptors in differentiated NG cells may be due to changes in the relative amount of the 5-HT3B subunit, particularly the ratio and distribution of the 5-HT3A to 3B subunits.