The earliest Pleistocene fossil forest of Dunarobba (Umbria, Italy) consists of a set of more than 70 tree trunks of an extinct species of sequoia or cypress with original cellulose still preserved. Spectral analyses of tree-ring series (325 and 448 yr in duration) combined with oxygen isotope analyses of the cellulose provide a glimpse into the mean annual temperature and the interannual climate variability that characterized this region at the beginning of the Pleistocene, when the concentration of atmospheric CO₂ was ~400 ppm. The high-frequency variability of the ring width time series shows significant spectral components that are consistent with the influence from the North Atlantic Oscillation, and to a lesser extent, solar cycles and El Niño–Southern Oscillation. The mean annual temperature estimate of ~19 °C, based on a model that combines ring widths and oxygen isotope values, is a full 6 °C warmer than the present-day value for this region. These elevated temperatures are consistent with estimates from pollen analyses and with estimates from higher latitudes.