Transmission electron microscopy was used to determine if eastern equine encephalitis (EEE) virus infects and replicates in the salivary glands of Culiseta melanura after 3 days of extrinsic incubation (EI). The Cs. melanura studied were from a colony strain, were orally infected, and had EI periods of 55–69 hours. Both naked nucleocapsids and enveloped virions were present in aggregates, suggestive of viral replication, within salivary gland acinar cells. Nucleocapsids were present in the cytoplasm below the plasma membrane that lined apical cavities. Enveloped virions occurred in the salivary matrix within apical cavities. Some nucleocapsids appeared to be budding through the plasma membrane around apical cavities and maturing into infectious virions. These results suggest that Cs. melanura is capable of biological transmission of EEE virus after ≤ 3 days of EI.