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Cell-free extracts prepared from whole yeast cells carry out selective and accurate transcription, in vitro, of purified yeast class III genes. Both 5 S rRNA and tRNA genes are specifically transcribed by DNA-dependent RNA polymerase III present in these whole cell extracts. These extracts also appear to carry out nucleolytic processing of the in vitro synthesized transcripts. Optimal conditions for specific class III gene transcription in vitro are defined. Initial fractionation of the yeast extract has indicated that multiple chromatographically separable factors (fractions) are required, in addition to RNA polymerase III, for specific in vitro transcription of class III genes.