A uridine diphosphate(UDP)-glucose:glucan synthetase can be demonstrated in detached cotton fibers (Gossypium hirsutum L.) and in an isolated particulate fraction from such fibers. When assayed with detached fibers, the kinetics of the glucan synthetase activity with respect to variation in substrate concentration is complex and indicates activation of the enzyme by the substrate. Activity is stimulated by Ca(2+) or Mg(2+) and beta-linked glucosides; the effect of the beta-linked glucosides is to shift the range in which substrate activation occurs to lower concentrations of UDP-glucose. At concentrations of UDP-glucose below 50 mum, addition of uridine triphosphate, in addition to beta-linked glucoside, results in significant stimulation of activity. This effect can be explained by the conversion of uridine triphosphate to UDP-glucose by UDP-glucose pyrophosphorylase, thereby raising substrate concentration to the activating range. In detached fibers, glucan synthetase activity is high at all stages of fiber development. The properties of the glucan synthetase of the isolated particulate fraction closely resemble those of the enzyme assayed in detached fibers; however, in contrast to detached fibers, the ability to detect enzyme activity is more dependent on fiber age, showing maximal activity between 16 and 18 days postanthesis, coincident with the time of rapid onset of secondary wall cellulose deposition.