Activation T-DNA tagging was used to generate four cytokinin-independent (cyi1-4) tobacco cell lines. Plants regenerated from the mutant lines displayed similar phenotypes: reduced apical dominance, poorly developed roots, delayed growth and flowering, and male and female sterility. Tissue culture experiments demonstrated that the mutations in the different lines uncouple cell proliferation from the effects of both cytokinin and auxin. No significant increase of cytokinin or auxin was found in transgenic calli in comparison with untransformed callus. The functional plant sequence tagged in one of the mutant lines, cyi1, was used to isolate an active cDNA, cyi1a, that was able to trigger cytokinin- and auxin-independent protoplast division. Northern analysis shows that the transcript corresponding to cyi1a accumulates to high levels in the untransformed protoplasts shortly before the onset of cell division, and that these levels decrease when protoplasts reach maximum rates of cell division. A small putative open reading frame, starting with the first ATG in cyi1a and encoding a 22 amino acid peptide, has the same activity in tobacco protoplasts as the whole cDNA. This activity is destroyed by a frame shift mutation. Apparently cyi1a encodes a peptide which participates in the events downstream of a joint point of cytokinin and auxin action leading to cell division.