The kinetoplast DNA networks of Trypanosoma cruzi exist under two forms which have been studied by equilibrium density centrifugation in CsCl gradients containing ethidium bromide and by band sedimentation analysis. The relative proportion of the two forms has been measured and varies significantly between the exponential and stationary phase of growth, suggesting that one of these forms is a replicative intermediate. Both forms exhibit very high sedimentation coefficients. The sedimentation velocity ethidium titration was used to measure the superhelix density of the kinetoplast DNA after having established the validity of the method with in vitro closed DNA networks. The superhelix density of the native form of the kinetoplast DNA minicircles is very low and varies according to the physiological state of the trypanosomes. Furthermore, we observed a significant increase of the superhelix density of the kinetoplast DNA of trypanosomes grown in the presence of ethidium.