A study of CO2 in soil gas was conducted in a bare plot in the unsaturated zone (USZ) of Yatir Forest, northern Negev, Israel. In 2006, 6 tubes for sampling of soil gas were inserted into the USZ to depths of 30, 60, 90, 120, 200, and 240 cm. Profiles of soil gas in the USZ were collected from the tubes 5 times between October 2007 and September 2008. Measurements of the collected profiles of soil gas were of CO2 (ppm), δ13C (′), and Δ14C (′). At all times, the concentration of CO2 in the soil gas was higher than in the air at the surface (CO2 ≃ 400 ppm; δ13C ≃ −9′). The main source of the CO2 in soil gas is from biotic activity released through roots of trees and of seasonal plants close to the surface. In the winter, the CO2 concentrations were lowest (6000 ppm) and the δ13C was −20′. In the spring and through the summer, the CO2 concentration increased. It was estimated that the major source of CO2 is at ≃240 cm depth (δ13C ≃ −22′; CO2 ≃ 9000 ppm) or below. Above this level, the concentrations decrease and the δ13C (′) become more positive. The 14C values in the measured profile are all less than atmospheric and biotic 14C. It was deduced that biotic CO2 dissolves in porewater to form carbonic acid, which then dissolves secondary carbonate (δ13C ≃ −8′; 14C ≃ −900′) from the sediments of the USZ. With the 14C data, the subsequent release of CO2 into the soil gas was then estimated. The 14C data, supported by the 13C and CO2 data, also indicate a biotic source at the root zone, at about 90 cm depth.