In vitro testing is the first important step in the development of new biomaterials. The human foetal osteoblast cell line hFOB 1.19 is a very promising cell model; however, there are vast discrepancies in cultivation protocols, especially in the cultivation temperature and the presence of selection reagent, geneticin (G418). We intended to use hFOB 1.19 for the testing of Zn-based degradable metallic materials. However, the sensitivity of hFOB 1.19 to zinc ions has not yet been studied. Therefore, we compared the toxicity of zinc towards hFOB 1.19 under different conditions and compared it with that of the L929 mouse fibroblast cell line. We also tested the cytotoxicity of three types of Zn-based biomaterials in two types of media. The presence of G418 used as a selection reagent decreased the sensitivity of hFOB 1.19 to Zn²⁺. hFOB 1.19 were more sensitive to Zn²⁺ at elevated (restrictive) temperatures. hFOB 1.19 were less sensitive to Zn²⁺ than L929 (both as ZnCl₂ and extracts of alloys). Therefore, the appropriate cultivation conditions of hFOB 1.19 during biomaterial testing should be chosen with caution.