Toxicological studies have demonstrated that intermittent PTH1-34 treatment is associated with an increased incidence of osteosarcoma in Fischer 344 rats. Comet and micronucleus (MN) tests, standard methods to evaluate genotoxic potential of drugs, were used to detect DNA and chromosome breaks, respectively, after PTH1-34 treatment. MC3T3 cells, primary osteoblast calvarial cells, and human osteoblasts were treated with PTH1-34 (50 and 100 nM) for 6 h/day for 21 days to mimic intermittent administration. Genotoxic assays were performed at 6 h and 7, 14, and 21 days. Osteoblasts extracted from bone marrow of mice treated with daily subcutaneous PTH1-34 injections (20 and 40 μg/kg) for 10 weeks as well as Hep-2, HeLa, and Hep-G2 cells were also tested. We observed a significant increase in DNA lesions and MN prevalence in human and murine osteoblasts treated with PTH1-34 compared to controls (P < 0.01). The effect observed in vitro and confirmed in vivo was time- and dose-dependent. For nonosteoblastic Hep-2 and HeLa cells we observed increased DNA damage and MN prevalence only later in the course of the protocol, after 21 days of treatment (P < 0.01). In Hep-G2 cells intermittent PTH1-34 did not induce DNA damage or chromosome breaks. Our results demonstrated that intermittent PTH increases DNA and chromosome breaks in osteoblasts. This genotoxic effect is attenuated in nonosteoblastic cells, and the ability to induce DNA damage is lost in cells with detoxification properties (HepG2 cells) tested in vitro.