The ability of abscisic acid (ABA) and abscisic acid analogs to induce freezing tolerance in fall rye (Secale cereale cv Puma) seedlings grown at nonhardening temperatures was investigated. Analogs were constructed with systematic alterations at C-1 (acid replaced with methyl ester, aldehyde or alcohol), at C-4, C-5 (trans double bond replaced with a triple bond), and at C-2′, C-3′ (double bond replaced with a single bond so that the side chain and C-2′ methyl groups were cis). Freezing tolerance (LT50) was determined 3, 4 and 6 days after the first of two consecutive applications of chemical (100 µM) to either the leaves or roots. All analogs were more effective when applied to the plant roots than when applied to the leaves. ABA, acetylenic ABA and 2′,3′-dihydroacetylenic ABA decreased the LT50 from −3 °C (control) to −9 °C. Consistent structure-activity relationships were only detected following root application. No single functional group altered was absolutely required for activity. The effect of any given change to the molecule was modified by the presence of other functional groups. For example, substituting the double bond in the ring with a single bond decreased activity, but concomitant substitution of the trans double bond in the side chain with a triple bond restored activity. In general, analogs with a cis, trans side chain were more active initially but rapidly lost activity, whereas acetylenic analogs maintained or gained activity over the three sampling times. The application of gibberellin biosynthesis inhibitors (100 µM; tetcyclacis or mefluidide) did not increase freezing tolerance beyond that induced by ABA, either alone or in combination with ABA. It can be concluded that ABA and certain ABA analogs can induce limited freezing tolerance in whole rye seedlings, and partially substitute for low temperature acclimation.