UDP-glucose is the primary substrate for cellulose synthase, the enzyme that produces the main chemical constituent of cotton fiber. Two enzymes, UDP-glucose pyrophosphorylase (UDPGp, EC 220.127.116.11) and sucrose synthase (SuSy, EC 18.104.22.168) catalyze the synthesis of UDP-glucose. SuSy plays an important role in cellulose metabolism during rapid secondary cell wall biogenesis by providing UDP-glucose directly to cellulose synthase. The exact role of UDPGp is unclear, but UDPGp enzyme activity increases during the period of development when cotton fiber is synthesizing massive amounts of cellulose. The objective of this study was to begin elucidating the role of UDPGp by determining the temporal expression of UDPGp genes during fiber development and in other cotton tissues. The results demonstrate that cotton fibers, seeds, and leaves express the same UDPGp gene at various stages of development, and there is an increase in the steady state level of UDPGp mRNA concomitant with secondary cell wall biosynthesis. The UDPGp steady state mRNA level is reduced in leaves upon wounding, so it is unlikely this UDPGp gene plays a role in callose biosynthesis. The deduced open reading frame of this sequence is 48% identical to an UDPGp from Dictyostelium discoideum Raper. The 5′ end of an UDPGp gene expressed in fiber was isolated and the organization of two introns determined. Motifs potentially important in controlling gene expression were also identified.