During whole-cell recording of rat osteoblastic cells with high-Cl- internal solutions, 10 sec hyperpolarizing jumps from 0 mV induce a slow inward current relaxation, which is shown to be carried by hyperpolarization-activated Cl- channels. This relaxation increases and becomes faster with stronger hyperpolarizations. It is insensitive to Cs+ ions but is blocked in a voltage-dependent manner by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) 1 mM and is reduced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) 0.1 mM. Cd2+ ions are potent blockers of this current, blocking completely above 300 microM. The amplitude of the Cl- current activated by a given hyperpolarization increases during the first 10-20 min of whole-cell recording. This evolution and the fact that some recently cloned Cl- channels have been reported to be activated both by hyperpolarization and by external hyposmolarity led us to investigate the effects of external osmolarity. Reducing the external osmolarity induces a large Cl- current. However, this hyposmolarity-induced Cl- current and the hyperpolarization-activated Cl- current are shown to be distinct; 1,9-dideoxy forskolin selectively blocks the hyposmolarity-activated current. We show that the hyperpolarization-activated Cl- current is osmosensitive, but in an unusual way: it is reduced by external hyposmolarity and is increased by external hyperosmolarity. Furthermore, these modulations are more pronounced for small hyperpolarizations. The osmosensitivity of the hyperpolarization-activated Cl- current suggests a mechanosensitivity (activation by positive external pressure) that is likely to be physiologically important to bone cells.