Strigolactones (SLs) are a novel class of phytohormones that shape shoot and root architecture. Carlactone, the precursor of SLs, is synthesized from all-trans-β-carotene by the sequential action of three enzymes: the all-trans/9-cis-β-carotene isomerase DWARF27 (D27) that reversibly converts all-trans-β-carotene into 9-cis-β-carotene (shown only for the rice D27) and the carotenoid cleavage dioxygenases 7 and 8 (CCD7 and CCD8). Genomes of higher plants encode two D27 homologs (D27-like 1 and D27-like 2) with unknown function. Rice and Arabidopsis d27 mutants show less pronounced high tillering/more branching phenotype, compared to ccd7 or ccd8 mutants. This difference might be the result of functional redundancy caused by the presence of D27-like 1 and D27-like 2. In this study, we investigated the enzymatic and biological activity of the Arabidopsis D27 and the rice and Arabidopsis D27-like 1, using both in vivo and in vitro studies. Our results show that AtD27 is a true ortholog of the rice D27. Like SLs, the biosynthesis of ABA requires an all-trans/9-cis-isomerization reaction. However, the enzyme postulated for this activity is still elusive. Our enzymatic activity tests exclude a direct involvement of AtD27 in ABA biosynthesis. Nevertheless, Atd27 mutant shows low level of ABA, and analysis of AtD27 promoter