This paper describes studies of initiation of transcription by RNA polymerase I in vitro. The protocols take advantage of the observation that active transcription complexes precipitate when incubated with S100 extracts. The pellets contain less than 5% of the protein present in unfractionated extracts and are stable to centrifugal washing. This permits rapid manipulation of the reaction conditions and facilitates kinetic studies of aspects of the initiation reaction. An initiated complex has been defined which forms rapidly at 30 degrees C and is associated with formation of the first phosphodiester bond of nascent rRNA. Once formed, initiated complexes are capable of elongation in the presence of heparin or KCl in concentrations sufficient to preclude subsequent initiation. One can therefore estimate the number of initiated complexes formed in a given reaction by measuring the number of full length transcripts recovered in a KCl or heparin-start reaction. The number of such complexes formed correlates well with the formation of a presumptive initiator trinucleotide ApCpU.