Because the hyperpolarization-activated cation-selective current Ih makes important contributions to neural excitability, we examined its long-term regulation by vitronectin, an extracellular matrix component commonly elevated at injury sites and detected immunochemically in activated microglia. Focusing on mouse hippocampal pyramidal neurons in organotypic slice cultures established at postnatal day 0 or 1 and examined after 3-4 days in vitro, we observed differences in the amplitude and activation rate of Ih between neurons in naïve and vitronectin-exposed slices (10 µg/ml added to serum-free medium), and between neurons in slices derived from wild-type and vitronectin-deficient mice. The potassium inward rectifier IK(ir), activated at similar voltages to Ih, was not affected by vitronectin. In CA1, differences in Ih amplitude primarily reflected changes in maximum conductance (Gmax): a 23.3% increase to 3.18 ± 0.64 nS from 2.58 ± 0.96 nS (p < 0.05) in vitronectin-exposed neurons, and a 17.9% decrease to 2.24 ± 0.26 nS from 2.73 ± 0.64 nS (p < 0.05) in neurons from vitronectin-deficient slices. The voltage of one-half maximum activation (V1/2) was not significantly affected by vitronectin exposure (-78.1 ± 2.3 mV vs. -80.0 ± 4.9 mV in naïve neurons; p > 0.05) or vitronectin deficiency (-83.8 ± 3.1 mV vs. -82.0 ± 2.9 mV in wild-type neurons; p > 0.05). In CA3 neurons, changes in Ih reflected differences in both Gmax and V1/2: in vitronectin-exposed neurons a 35.4% increase in Gmax to 1.30 ± 0.49 nS from 0.96 ± 0.26 nS (p < 0.01), and a +3.0 mV shift in V1/2 to -89.8 mV from -92.8 mV (p < 0.05). The time course of Ih activation could be fit by the sum of two exponential functions, fast and slow. In both CA1 and CA3 neurons the fast component amplitude was preferentially sensitive to vitronectin, with its relatively larger contribution to total current in vitronectin-exposed cells contributing to the acceleration of Ih activation. Further, HCN1 immunoreactivity appeared elevated in vitronectin-exposed slices, while HCN2 levels appeared unaltered. We suggest that vitronectin-stimulated increases in Ih may potentially affect excitability under pathological conditions.