In this study, male fathead minnows (FHM) (Pimephales promelas) and juvenile rainbow trout (RT; Oncorhynchus mykiss) were exposed to two different surfactant mixtures of analytical-grade nonlyphenol, 4-tert octyphenol, octylphenol ethoxylates, nonylphenol ethoxylates, and the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). After a 7-days exposure to environmentally relevant concentrations of these compounds, there was no difference in the relative mRNA expression of vitellogenin (VTG) in male juvenile RT exposed to individual compounds or the 2,4-D-surfactant mixture compared with the control. In male FHM, there was a significant increase in VTG mRNA expression in the high individual treatments of 2,4-D and the surfactants but not the 2,4-D-surfactant mixtures compared with the control. These results were compared with another study exposing male FHM to individual and a mixture of alkylphenols and alkylphenol ethoxylates in two different combinations with the herbicide diuron and the insecticide bifenthrin. There were no differences in the relative expression of VTG mRNA amongst individual exposures and the control. Interestingly, when the ethoxylate mixture was combined with diuron, there was a significant decrease in the relative mRNA expression of VTG compared with the control. However, when the ethoxylate mixture was combined with both diuron and bifenthrin, there was a significant increase in the relative mRNA expression of VTG in male compared with all other groups in the multichemical mixture. The results of this study highlight differences between species and measurements of VTG in assessing the risk of mixtures to aquatic organisms.