OBJECTIVE: Titanium tetrafluoride (TiF4) has shown promising effect in preventing tooth lesions. Therefore, we compared the cytotoxicity of TiF4 with sodium fluoride (NaF) (already applied in Dentistry) considering different fluoride concentrations, pH values and experimental models. MATERIALS AND METHODS: Step 1) NIH/3T3 fibroblasts were exposed to mediums containing NaF or TiF4 (from 0.15 to 2.45% F), both at native and adjusted pH, for 6 h. Step 2) NIH/3T3 were exposed to NaF or TiF4 varnishes with 0.95, 1.95 or 2.45% F (native pH), for 6, 12 or 24 h. We applied MTT (1st and 2nd steps) and Hoescht/PI stain (2nd step) assays. Step 3) NIH/3T3 were exposed to NaF or TiF4 varnish (2.45% F), at native pH, for 6 or 12 h. The cell stiffness was measured by atomic force microscopy (AFM). RESULTS: Step 1) All cells exposed to NaF or TiF4 mediums died, regardless of the F concentration and pH. Step 2) Both varnishes, at 1.90 and 2.45% F, reduced cell viability by similar extents (33-86% at 6 h, 35-93% at 12 h, and 87-98% at 24 h) compared with control, regardless of the type of fluoride. Varnishes with 0.95% F did not differ from control. Step 3) TiF4 and NaF reduced cell stiffness to a similar extent, but only TiF4 differed from control at 6 h. CONCLUSIONS: Based on the results of the 3 experimental steps, we conclude that TiF4 and NaF have similar cytotoxicity. The cytotoxicity was dependent on F concentration and exposure time. This result gives support for testing the effect of TiF4 varnish in vivo.