Tracheary-element (TE) differentiation in suspension-cultured mesophyll cells of Zinnia elegans L. was completely inhibited by caffeine and theophylline only when these methylxanthines were applied at least 8 h prior to the appearance of secondary cell-wall thickenings. In contrast, the calcium-channel blocker nifedipine completely inhibited TE differentiation when applied only 2-3 h prior to the onset of secondary cell-wall deposition. This indicates the involvement of a methylxanthine-inhibitable event in TE differentiation that is distinguishable from an event dependent on influx of extracellular calcium. The correlation between the time of appearance of chlorotetracycline fluorescence (an indicator of sequestered Ca(2+)) and loss of methylxanthine effectiveness indicates that inhibition by methylxanthines may result from release of Ca(2+) from intracellular stores. Methylxanthines with high potencies against adenosine 3' ∶ 5'-cyclic monophosphate (cAMP) phosphodiesterase and adenosine receptors were less effective inhibitors of TE differentiation, indicating that inhibition of differentiation by methylxanthines is independent of cAMP metabolism. The role of cAMP in transduction of the cytokinin signal, which was proposed previously on the basis of stimulation of TE differentiation by theophylline, was investigated using the non-hydrolyzable analog 8-bromo-cAMP. Although 8-bromo-cAMP stimulated differentiation in the absence of inductive concentrations of cytokinin, the non-cyclic analog 8-bromo-AMP was even more effective, indicating that 8-bromo-cAMP behaves as a cytokinin analog, rather than a second messenger, in stimulating TE differentiation.