Objective. This study investigated the long-term cytotoxic response of thermoplastic polyamide and conventional polymethyle metacrylate (PMMA) denture base materials. Materials and ethods. Twenty discs were prepared for each polyamide, heat and cold cured PMMA denture base resins (totally 60) and divided into four sub-groups (n = 5). Cytotoxicity was assessed with the direct cell contact method using cell viability and neutral red (NR) uptake assay. Each sub-group was tested at initial and after being aged for 24 h, 1 week and 8 weeks with artificial saliva according to ISO 10993 standards. Results. There were no significantly difference among the materials and control groups after initial, 24 h and 1 week testing. In 24 h testing, only Deflex was more toxic according to the Control group (p < 0.05). After 8 weeks of aging with artificial saliva, all materials were significantly cytotoxic when compared to the control group. QC20 was more toxic than Deflex and SC Cold Cure (p < 0.05). There were significant differences between the 8 week aging group and the initial, 24 h and 1 week testing for all materials (p < 0.05). Conclusions. Cytotoxicity of all tested denture base materials increased significantly after the long-term aging. Therefore, long-term aging may be useful to determine a dental material's toxicity. Polyamide denture base material had a similar toxicity profile with conventional heat-and cold-cured PMMA.