Grid cells in medial entorhinal cortex (MEC) fire in spatially specific locations in a repeating hexagonal grid, with smaller spacing between grid fields for neurons in more dorsal anatomical locations and larger spacing between grid fields for neurons in more ventral anatomical locations (Hafting et al., 2005; Sargolini et al., 2006). Single cell recordings using whole-cell patch clamp techniques demonstrated a correlation between intrinsic properties dependent on the hyperpolarization activated cation current (Ih) and the topographical organization of grid cell field spacing along the dorsal-ventral axis of MEC (Giocomo et al., 2007; Giocomo & Hasselmo, 2008). In slice recordings, knockout of the HCN1 subunit which conducts Ih reduces the dorsal-ventral gradients in intrinsic properties (Giocomo & Hasselmo, 2009). To directly test the role of Ih and intrinsic properties in determining the properties of the grid cell field spacing topography along the dorsal-ventral axis in vivo, we recorded grid cells along the dorsal-ventral axis of layer II/III MEC in forebrain restricted HCN1 knockout mice and matched wild type littermates. We found a significant increase in grid cell field spacing in HCN1 knockout mice as well as complementary changes in the intrinsic firing properties of grid cells.