Larval topsmelt (Atherinops affinis) and inland silversides (Menidia beryllina) were exposed in 96‐h static acute toxicity tests to 11 chemicals to determine the relative sensitivity of the two atherinid species. High to low LC50 ratios for endosulfan, methoxychlor, carbophenothion, chlorpyrifos, terbufos, fenvalerate, permethrin, 4‐nitrophenol, and sodium lauryl sulfate were within a factor of <2 for the two species. Atherinops affinis was more sensitive to both azinphos‐methyl and 2,4‐dinitrophenol by factors of 6.7 and 4.4, respectively. Comparisons of the relative sensitivity of Atherinops affinis with three freshwater fish species (Lepomis macrochirus, Oncorhynchus mykiss, Pimephales promelas) and one estuarine fish species (Cyprinodon variegatus) are presented. Sensitivities were similar between Atherinops affinis and the two most sensitive freshwater species, Lepomis macrochirus and Oncorhynchus mykiss. Atherinops affinis is easily transported, cultured, and maintained in the laboratory and readily adaptable for use in toxicological studies.