The aggregation pattern of penicillin V (PCV) in a 0.15 M potassium chloride solution at 298.2 K is investigated by frontal derivative chromatography on Sephadex G-10 columns and is quantitatively analyzed on the basis of a stepwise aggregation model and a trimer-dodecamer model. The aggregation parameters for these models are best fitted to the observed centroid volume data. The derivative chromatogram of PCV is simulated on the basis of plate theory with the aggregation models. The height and position of the peak simulated on the basis of the stepwise aggregation model are in excellent agreement with the observed ones, whereas those simulated on the basis of the trimer-dodecamer model remarkably differ from the observed ones, particularly in dilute solutions. Thus, PCV forms dimer and self-associates stepwise.