Summary 1 Bacteria are common and important constituents of aquatic ecosystems but little is known about their populations; most measurements commonly employed are at a grosser, assemblage-level. The purpose of this study was to compare longitudinal patterns in microbial assemblages and populations along a river ecosystem. 2 Water samples were collected under base flow conditions from 13 main stem and four tributary sites along the Ogeechee River spanning the piedmont and coastal plain of Georgia, USA. Based on the detection of active cells with abundant ribosomes, two species of bacteria, Acinetobacter calcoaceticus and Pseudomonas putida, represented 0.4–4.9% of individuals in the Domain Bacteria. The abundance of P. putida was low in the headwater region of the river, perhaps because of lower concentrations of aromatic compounds. 3 The abundance of members of the Domain Bacteria peaked in the upper and lower orders of the river and were low in the mid-orders, where protozoa were numerous. 4 Higher DOC quality (based on bacterial growth) in the headwaters and peak protozoa numbers in the mid-section of the river, may have contributed to the spatial patterns observed in the bacterial assemblage. Results suggest that there is a longitudinal pattern of changing relationships between labile DOC, protozoa and bacteria but that, within the bacterial assemblage, species differ in their responses.