The spatial and temporal distributions of decapod crustacean and juvenile fish species in the Ashepoo, Combahee, and South Edisto (ACE) rivers in the ACE Basin National Estuarine Research Reserve were examined from 1993 to 1999. Nekton samples were collected monthly during slack low to early flood tide by bottom trawl from 12 fixed stations (four stations/river) along the salinity gradient in the reserve. During the 6-year survey, 79 species of fish and 26 decapod crustacean species were caught. Coastal marine species represented more than 80% of the species collected during the survey; the remaining finfish were permanent residents, freshwater, and diadromous species. Star drum (Stellifer lanceolatus), Atlantic croaker (Micropogonias undulatus), bay anchovy (Anchoa mitchilli), and spot (Leiostomus xanthurus) constituted >68% of the total number of individual fishes collected. White shrimp (Litopenaeus setiferus) and brown shrimp (Farfantepenaeus aztecus) constituted ∼87% of the total number of individual decapods collected. Fish and decapod crustacean assemblage structure in the three rivers were analyzed for spatial and seasonal patterns. Spatial distribution of the species assemblages in the estuarine systems appeared to be strongly influenced by the physiological tolerances of the individuals to salinity gradients in the study area. Seasonal variations in species diversity and abundance appeared to be related to migration and recruitment of species to the estuarine system, and there were two annual recruitment cycles: winter–spring (October to March) and summer–fall (April to September). The variations in species diversity among the stations in the mesohaline zone were driven by the abundance of the dominant species.