Contaminant exposure is one possible contributor to population declines of endangered fish species in the Sacramento–San Joaquin Estuary, California, including the endangered delta smelt (Hypomesus transpacificus). Herein we investigated transcriptional responses in larval delta smelt resulting from exposure to water samples collected at the Department of Water Resources Field Station at Hood, a site of concern, situated upstream of known delta smelt habitat and spawning sites and downstream of the Sacramento Regional Wastewater Treatment Plant (SRWTP). Microarray assessments indicate impacts on energy metabolism, DNA repair mechanisms and RNA processing, the immune system, development and muscle function. Transcription responses of fish exposed to water samples from Hood were compared with exposures to 9% effluent samples from SRWTP, water from the Sacramento River at Garcia Bend (SRGB), upstream of the effluent discharge, and SRGB water spiked with 2 mg/L total ammonium (9% effluent equivalent). Results indicate that transcriptomic profiles from Hood are similar to 9% SRWTP effluent and ammonium spiked SRGB water, but significantly different from SRGB. SRGB samples however were also significantly different from laboratory controls, suggesting that SRWTP effluent is not solely responsible for the responses determined at Hood, that ammonium exposure likely enhances the effect of multiple-contaminant exposures, and that the observed mortality at Hood is due to the combination of both effluent discharge and contaminants arising from upstream of the tested sites.