OBJECTIVE: Bioelectrical impedance analysis (BIA) of hydration and body composition has made significant progress during the past 3 decades. With the development of Bioimpedance spectroscopy (BIS), bioimpedance has been expanded to reliably predict extracellular fluid (ECF) and total body water (TBW), allowing the calculation of fat-free mass (FFM) and fat mass (FM). In this study, a new BIS device (ImpediVet™), designed for body composition measurements in animals, was assessed for precision and accuracy in measuring TBW, FFM and FM in rats. METHODS: In a validation study, 25 rats were measured for body composition (TBW, FFM and FM) using BIS and chemical carcass analysis (CCA). BIS precision was assessed by the coefficient of variation using multiple BIS readings, while BIS accuracy was assessed by regression analysis of BIS and CCA values for each body compartment. In a cross-validation study, prediction equations generated from the validation group for TBW, FFM and FM were applied to an independent cohort of 25 rats that were measured by BIS and CCA. Linear regression analysis and paired t-tests were used to assess significance of relationships and measurement differences within groups. RESULTS: In the validation study, BIS was highly correlated with CCA for TBW (r(2)=0.988), FFM (r(2)=0.987) and FM (r(2)=0.966). Even so, BIS significantly underestimated TBW (mean: -31.07 g, -13.3%, p<0.001) and FFM (-50.69 g, -15.5%, p<0.001), while overestimating FM (+65.75 g, +63.5%, p<0.001). In the independent, cross-validation group of rats the prediction equations accurately predicted carcass values for TBW (-0.2%, p=0.350), FFM (-0.2%, p=0.457) and FM (+1.5%, p=0.508). CONCLUSION: Based on these results, BIS provided a precise and accurate means to determine in vivo body composition in rats.