The yeast cytoplasmically inherited genetic determinant [PSI+] is presumed to be a manifestation of the prion-like properties of the Sup35 protein (Sup35p). Here, cell-free conversion of Sup35p from [psi(-)] cells (Sup35(psi-)) to the prion-like [PSI+]-specific form (Sup35p(PSI+)) was observed. The conversion reaction could be repeated for several consecutive cycles, thus modeling in vitro continuous [PSI+] propagation. Size fractionation of lysates of [PSI+] cells demonstrated that the converting activity was associated solely with Sup35p(PSI+) aggregates, which agrees with the nucleation model for [PSI+] propagation. Sup35p(PSI+) was purified and showed high conversion activity, thus confirming the prion hypothesis for Sup35p.