Physiologic systems can be represented by compartmental models which describe the uptake of radio-labeled tracers from blood to tissue and their subsequent washout. Arterial and venous time-activity curves from isolated heart experiments are analyzed using spectral analysis, in which the impulse response function is represented by a sum of decaying exponentials. Resolution and uniqueness tests are conducted by synthesizing isolated heart data with predefined compartmental models, adding noise, and applying the spectral analysis technique. Venous time-activity curves are generated by convolving a typical arterial input function with the predefined spectrum. The coefficients of a set of decaying exponential basis functions are determined using a non-negative least squares algorithm, and results are compared with the predefined spectrum. The uniqueness of spectral method solutions is investigated by computing model covariance matrices, using error propagation and prior knowledge of noise distributions. Coupling between model parameters is illustrated with correlation matrices.