The self-association of penicillin V in aqueous solution at 298.15 K has been examined by conductivity, density, ultrasound, static light scattering, and microcalorimetry techniques. Two critical concentrations were detected in conductivity, light scattering, and ultrasound data over the concentration range 0-0.35 mol kg(-1). Light scattering measurements indicate the formation of trimers at the first critical concentration (0.04 mol kg(-1)) and subsequent formation of aggregates of aggregation number 12 at the second critical concentration (0.23 mol kg(-1)). Analysis of the apparent molar volumes and isentropic apparent molar compressibilities has also provided evidence for limited association at concentrations below the first critical concentration. A model of the association process and possible structures of the primary and secondary aggregates have been proposed on the basis of an analysis of the thermodynamics of the self-association.