Free and conjugated abscisic acid (ABA) levels in stem‐cultured plantlets of potato (Solanum commersoniiDun, PI 458317) during cold acclimation were measured. The levels of free and conjugated ABA were measured by an enzyme immunoassay (EIA) with rabbit anti‐ABA‐serum. The use of immunoglobulin G fraction purified from rabbit antiserum and the methylated form of ABA resulted in an improved measuring range (0.01 to 10 pmol ABA) and precision (slope of logit‐log plot, −1.35) of EIA, compared to the use of antiserum and free ABA. Estimates of the EIA were consistent with those resulting from a commercial EIA. Under a 4/2°C (day/night) temperature regime, the potato plantlets increased cold hardiness from −5°C (warm‐grown control) to −10°C by the 7th day. During the same period, there were two transitory increases in free ABA, the first one three‐fold from 1.5 to 5.3 nmol (g dry weight)−1on the 2nd day and the second one five‐fold from 1.5 to 7.6 nmol (g dry weight)−1on the 6th day. Each increase in ABA concentration was followed by an increase in cold hardiness. There was no significant change in conjugated ABA content (4.2±0.6 nmol [g dry weight]−1) throughout the cold acclimation period. The lack of an interrelationship between levels of free and conjugated ABA suggested that the transitory increase in free ABA during cold acclimation was not a result of the conversion of conjugated ABA. The increase in free ABA due to biosynthesis of ABA during potato