Three steps have been established during the Holocene formation of the bar-built estuary of Las Nuevas (Doñana National Park, Spain), on the basis of the clay mineralogy variations and the palaeontological record. The first step is characterized by the presence of ostracodes and homogeneous quantities of clay minerals (17–20% illite, 25–29% smectites), values of smectite (0.64–0.70) and illite (0.60–0.70) crystallinity indexes, and the ratio of AlVI/(FeVI + MgVI) in illite (0.46–0.47). This zone is interpreted as a very shallow lagoon with euryhaline conditions. The presence of roots, the progressive disappearance of foraminifers and an increase in the smectite content (up to 35%) define the second step. A salt-marsh environment with low-energy hydrodynamic conditions is deduced for this zone. The third step is characterized by an increase in illite content (up to 35%), and a decrease of the smectite content (up to 21%). The smectite crystallinity index decreased to 0.38, whereas the illite ratio AlVI/(FeVI + MgVI) decreased to 0.36. In this zone, the ostracode assemblage contains numerous juvenile stages of coastal species coinciding with lumachelle accumulations of the estuarine bivalves, abundant foraminifers and the presence of charophytes. This zone represents a strong marine input, probably caused by storms.