The internal concentration represented by the critical body residue (CBR) is an ideal indicator to reflect the intrinsic toxicity of a chemical. Whilst some studies have been performed on CBR, the effect of exposure route on internal toxicity has not been investigated for fish. In this paper, acute toxicity data to fish comprising LC50 and LD50 values were used to investigate CBR. The results showed that exposure route can significantly affect the internal concentration. LD50 and CBR calculated from LC50 and BCF both vary independently of hydrophobicity as expressed by log Kow; conversely, LC50 is related to log Kow. A poor relationship was observed between LC50 and LD50, but the relationship can be improved significantly by introduction of log Kow because log CBR is positively related to log LD50. The parallel relationship of log CBR-log Kow and log LD50-log Kow indicates that LD50 does not reflect the actual internal concentration. The average LD50 is close to the average CBR for less inert and reactive compounds, but greater than the average CBR for baseline compounds. This difference is due to the lipid fraction being the major storage site for most of the baseline compounds. Investigation on the calculated and observed CBRs shows that calculated CBRs are close to observed CBRs for most of compounds. However, systemic deviations of calculated CBRs have been observed for some compounds. The reasons for these systemic deviations may be attributed to BCF, equilibrium time and experimental error of LC50. These factors are important and should be considered in the calculation of CBRs.