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The suggested link between intracellular cytokinin signaling and phospholipase D (PLD, EC 3.1.4.4.) activity (Romanov et al. 2000, 2002) was investigated. The activity of PLD in the early period of cytokinin action was studied in vivo in derooted Amaranthus caudatus seedlings, using the level of phosphatidylbutanol production as a measure of PLD activity. Rapid activation of phosphatidylbutanol synthesis was demonstrated as early as within 5 min of cytokinin administration. Neomycin, a known phosphatidylinositol‐4,5‐bisphosphate (PIP₂) antagonist, strongly repressed both physiological cytokinin effect and cytokinin‐dependent PLD activation. N‐acylethanolamine (NAE 12), an inhibitor of α‐class PLD, did not influence significantly cytokinin effect on Amaranthus seedlings. Together, results suggest the involvement of PIP₂‐dependent non‐class α‐PLD in the molecular mechanism of cytokinin action.