In addition to the reported single cDNA sequence encoding vitellogenin in the Pacific oyster Crassostrea gigas (referred as Cg-Vtg1), we isolated and characterized the 3′ end of a novel vitellogenin (Vtg) in C. gigas, named Cg-Vtg2. The transcript size is up to 6 kb long and includes a D-type von Willebrand domain, characteristic of most vitellogenins, but not occurring in Cg-Vtg1. The expression of Cg-Vtg2 was found restricted to follicular cells in maturing and ripe female gonads. Quantitative expression analysis of both vitellogenins showed a closely correlated transcription pattern during the female gametogenic cycle, with the highest expression during the ripe (mature) stage, and a declining expression, to almost undetectable levels, in post-spawning (resting) stage. Levels of vitellogenin mRNA associates with the quantity of ingested food. Mature female oysters fed either a 2% or a 12% algal biomass per female biomass, showed a 2.9-fold increase in expression of Cg-Vtg1 and a 3.7-fold increase of Cg-Vtg2 expression levels in those females fed the higher level of algae, suggesting that Vtg's could be used as quantitative indicator of vitellogenesis. Our results suggest that vitellogenins are synthesized during the late period of vitellogenesis, when most of the yolk has already been incorporated into maturing oocytes.