In colloidal systems physical-chemical changes are often a function of volume fraction and sample dilutions are critical. While most methods to characterize colloidal particles either require dilution or some disruption, acoustic spectroscopy can be performed in situ, without dilution. Objective of this work was to determine the effects of concentration, heating and acidification on the acoustic and electroacoustic properties of casein micelles in skim milk. The ultrasonic attenuation of skim milk increased with concentration of milk and frequency, and the average size of the colloidal particles calculated from the frequency dependence of attenuation was about 0.15 mu m for both unheated and heated milk. When milk was concentrated by ultrafiltration, at 3x and 4x concentration (based on volume reduction), the calculated size deviated from that derived in undiluted or mildly concentrated milk, most likely because of increased particle-particle interactions. Electroacoustic measurements revealed a constant dynamic mobility of the particles in undiluted and concentrated milk, while lower mobilities were observed for milk diluted in permeate. The c-potential measured was significantly higher than the values measured using dynamic light scattering, with a value of -45.8 mV for casein micelles in unheated milk. With acidification, the c-potential decreased monotonically. Heating profoundly affected the change in charge with pH of the micelles, and it was concluded that the interaction of casein micelles with the whey proteins increased the surface charge of the casein micelles. (C) 2010 Elsevier Inc. All rights reserved.