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Statement of problem. Occlusal devices can be either conventionally processed, milled, or printed. However, little is known about the biocompatibility of 3D printing resin materials.Purpose. The purpose of this in vitro study was to compare the viability and morphology of human gingival fibroblast cells (HFG-1) after cultivation on conventionally processed, milled, and printed occlusal device materials with different surface treatments. Material and methods. Disks of a conventionally processed (PalaXpress Clear [pP]), milled (Yamahachi PMMA Clear [sY]), and 2 different printed materials (Dental LT Clear Resin [aD];Freeprint splint [aF]) were prepared. The surfaces of the specimens were finished by using 2 different treatments (unpolished and polished with P1200-grit silicon carbide paper). HGF-1 cells were cultivated on the specimens for 24 hours, and a viability assay was performed by using polystyrene disks as a control (n=9 disks per group). Cell morphology and the topography of the specimens were examined with scanning electron microscopy (n=3 disks per group). Two-way analysis of variance was applied to determine the effect of material and surface treatment followed by the post hoc Fisher least significant difference test (a=.05).Results. Overall, material (P<.001) and surface treatment (P<.001) significantly influenced the viability of HGF-1 cells. The viability of cells on all specimens displayed mean values between 0.85 and 1.01 compared with the control except for unpolished aD (0.00 ±0.07) and aF (0.02 ±0.05) that had only a few cells with a round shape.Conclusions. The behavior of HGF-1 cells on conventionally processed and milled specimens was similar and not dependent on the surface treatment. Unpolished printed specimens had a cytotoxic effect. However, after polishing, cell behavior was similar to that of the conventionally processed and milled specimens.