The fish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, but concerns have been raised for its predictivity given that a few compounds have been shown to exhibit a weak acute toxicity in the fish embryo. In order to better define the applicability domain and improve the predictive capacity of the fish embryo test, we performed a systematic analysis of existing fish embryo and acute fish toxicity data. A correlation analysis of a total of 153 compounds identified 28 compounds with a weaker or no toxicity in the fish embryo test. Eleven of these compounds exhibited a neurotoxic mode of action. We selected a subset of eight compounds with weaker or no embryo toxicity (cyanazine, picloram, aldicarb, azinphos-methyl, dieldrin, diquat dibromide, endosulfan, and esfenvalerate) to study toxicokinetics and a neurotoxic mode of action as potential reasons for the deviating fish embryo toxicity. Published fish embryo LC₅₀ values were confirmed by experimental analysis of zebrafish embryo LC₅₀ according to OECD guideline 236. Except for diquat dibromide, internal concentration analysis did not indicate a potential relation of the low sensitivity of fish embryos to a limited uptake of the compounds. Analysis of locomotor activity of diquat dibromide and the neurotoxic compounds in 98 hpf embryos (exposed for 96 h) indicated a specific effect on behavior (embryonic movement) for the neurotoxic compounds. The EC₅₀s of behavior for neurotoxic compounds were close to the acute fish toxicity LC₅₀. Our data provided the first evidence that the applicability domain of the fish embryo test (LC₅₀s determination) may exclude neurotoxic compounds. However, neurotoxic compounds could be identified by changes in embryonic locomotion. Although a quantitative prediction of acute fish toxicity LC₅₀ using behavioral assays in fish embryos may not yet be possible, the identification of neurotoxicity could trigger the conduction of a conventional fish acute toxicity test or application of assessment factors while considering the very good fish embryo–acute fish toxicity correlation for other compounds.