In this paper, we analyze the pattern of distribution of three populations of picoplanktonic autotrophs measured using flow cytometry. Prochlorococcus sp., Synechococcus sp. and eukaryotic picoplankton were investigated at 58 stations to the south of the Iberian Peninsula including the northeastern Gulf of Cádiz, Strait of Gibraltar and northwestern Alborán Sea, during July 1995. There were important differences in the oceanographic conditions in these regions, with warmer, low-nutrient and low-chlorophyll waters in the Atlantic sector and colder, high-nutrient and high-chlorophyll waters in the Alborán sector in which upwelling occurred. The biomass concentration of picoplanktonic cells was two times higher in the Atlantic area than in the richer Alborán Sea waters. The average integrated abundance of picophytoplankton was negatively correlated with chlorophyll a concentration as well as with inorganic nitrogen availability. Prochlorococcus and Synechococcus were the most abundant groups, and they showed a complementarity in their spatial distribution, with a shallower and more coastal distribution of Synechococcus and the presence of Prochlorococcus mainly at offshore stations and at deeper depths. The ratio of autotrophic picoplankton/total autotrophic biomass showed significant differences on both sides of the Strait, with much higher values in the Gulf of Cádiz sector.