Aim Cross-system comparisons of species richness of benthic macrofauna and environmental factors were made of estuaries and adjacent sea areas in order to reveal possible regulating factors of estuarine biodiversity.Location Denmark.Methods Annual species abundance and biomass data from four years, from unvegetated soft sea/estuary floors were used from 26 grids, of which 15 were situated in estuaries. Bottom water data for oxygen concentration, salinity, and temperature were obtained from stations in or close to, the grids. Data on nutrient loading, water residence time and morphology were obtained for whole estuaries. Species richness from the grids, standardized to the same sample size, were related to environmental variables using linear regression.Results Species richness was unrelated to oxygen deficiency and productivity, but positively related to salinity. However, an equally high degree of explanation ( R 2 ∼ 0.70) was obtained using a model where richness was positively related to saltwater flux, which was computed from estuary volume and residence time and corrected for freshwater flux. The relationship was present for the dominating groups with pelagic dispersal, Annelida and Mollusca, but not for Crustacea, where recruitment is mainly by benthic pathways. Saltwater flux was strongly positively correlated with salinity, illustrating the high importance of flux from adjacent sea areas for water renewal in Danish estuaries. Similarity of species composition was greater for sites within than between saltwater current pathways, and the increases of richness with saltwater flux and salinity in the estuaries was largely due to marine species occurring in the open sea areas.Conclusion Danish estuaries are largely open systems where a part of estuarine species richness is sustained by dispersal from a species pool in adjacent seas. Results are consistent with the barrier prediction of island biogeography theory, but not the island size prediction.