The cuttlefish (Sepia officinalis) is the most important traditional fishing activity in the Sado estuary. In order to analyse the spatial and temporal distribution patterns and to study the reproductive cycle of this species, twenty sampling areas were towed along the estuary on a monthly basis from May 2001 to April 2002. The higher densities were obtained in summer and autumn in the lower and southern area of the estuary while spring showed the higher biomass. Mature individuals prevailed in the inner zone of the estuary during spring while juveniles were dominant in autumn and concentrated in the lower part of the estuary. Almost all individuals leave the estuary during winter. The weight–length relationship was significantly different according to sex, being females heavier than males for individuals larger than 106 mm. Sex-ratio was similar to 1:1 in every season. Females had high values of gonadosomatic index during the spawning season with a major peak in June, while males showed similar values throughout the spawning season. The length at first maturity was estimated at 80 mm mantle length (ML) for females and 59 mm ML for males. In the Sado estuary only one generation of cuttlefish breeders were found with an average of 110 mm ML for males and 122 mm ML for females, which is consistent with breeders in their first year.