Mean layer virtual temperature estimates, based on geopotential height measurements, form the basis for one approach being used to monitor changes in upper-air temperature. However, virtual temperature is a function of atmospheric moisture content as well as temperature. This paper investigates the impact of real or apparent changes in atmospheric moisture on changes in mean layer virtual temperature. Real changes in mean layer specific humidity of up to 50% would cause changes in mean layer virtual temperature of less than 1°C, except in the tropical boundary layer, where the high moisture content would lead to larger virtual temperature changes. The effect of humidity changes is negligible in polar regions and most pronounced in the tropics, which could influence the interpretation of the latitudinal gradient of virtual temperature trend estimates. Improvements in radiosonde humidity sensors since 1958 have led to an apparent decrease in atmospheric humidity. On global average, for the 850–300-mb layer, such changes are estimated to contribute to an apparent cooling of between 0.05° and 0.1°C, or about 10% to 20% of the observed warming trend since 1958.