Abstract: 5S rRNA genes from Saccharomyces cerevisiae were examined by Miller chromatin spreading, representing the first quantitative analysis of RNA polymerase III genes in situ by electron microscopy. These very short genes, approximately 132 nt, were engaged by one to three RNA polymerases. Analysis in different growth conditions and in strains with a 4-fold range in gene copy number revealed regulation at two levels: number of active genes and polymerase loading per gene. Repressive growth conditions (rapamycin or post-exponential growth) led first to fewer active genes, followed by lower polymerase loading per active gene. Pol III elongation rate was estimated in the range of 60-75 nt/sec with a reinitiation interval of approximately 1.2 sec. The yeast La protein, Lhp1, was associated with 5S genes. Its absence had no discernible effect on amount or size of 5S RNA produced, yet resulted in more polymerases per gene on average, consistent with a non-rate limiting role for Lhp1 in a process such as polymerase release/recycling upon transcription termination.