The differentiation of secondary xylem fibers was studied in cultured hypocotyl segments of Helianthus annuus L. It is shown that cytokinin is both a limiting and controlling factor in the early stages of fiber differentiation. In the absence of kinetin or zeatin, there was no fiber differentiation. However, cytokinin could induce fiber differentiation only in the presence of indoleacetic and gibberellic acids. First fibers were observed in the tissue after 12 days in culture, and their number increased linearly during the following 2 weeks. At low cytokinin levels, there was a positive correlation between cytokinin concentration in the medium and the number of fibers formed in the explants. A similar correlation was also found at low gibberellic acid concentrations. At high concentration, zeatin was more effective than kinetin. It seems that later stages of fiber differentiation can occur in the absence of cytokinin. It is proposed that the mechanism which controls and determines the early stages of fiber differentiation is based on an interaction of three major hormonal signals: indoleacetic acid plus gibberellic acid from the leaves with zeatin from the root apices.