The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and the level of its mRNA have been determined at various stages of tomato fruit development. The HMGR reaction makes mevalonate, a necessary component in the synthesis of all isoprene containing compounds, such as sterols and carotenoids. A cDNA clone encoding the active site region of HMGR has been isolated from a tomato library derived from young-fruit mRNA. The clone hybridizes to a one- or two-copy fragment in high-stringency DNA gel blot analyses and detects an mRNA of approximately 3.0 kb. Both HMGR activity and mRNA levels are high in early stages of tomato fruit development, when rapid cell division occurs, as well as in the subsequent early stages of cellular expansion. In contrast, ripening fruit have very low levels of reductase activity and mRNA, even though large amounts of the carotenoid lycopene are synthesized during this period. Furthermore, in vivo inhibition of HMGR during early fruit stages disrupts subsequent development, whereas inhibition during later stages of fruit expansion has no apparent effect on ripening. We conclude that the pool of mevalonate responsible for the synthesis of phytosterols is synthesized primarily during the first half of tomato fruit development. In addition, the final period of fruit expansion and ripening is not dependent upon HMGR activity, but instead utilizes a preexisting pool of pathway intermediates or requires the use of salvage pathways in the cell.