In Tetrahymena thermophila, an “antisense ribosome” technology has been developed for inhibiting gene expression and generating novel mutants. Short segments of genes are inserted in antisense orientation into an rDNA vector in a region corresponding to an external loop of the folded rRNA. DNA segments derived from the 5′-ends of genes have proven most effective in reducing cognate gene expression. To investigate the efficacy of other genic regions, we generated Tetrahymena cell lines with antisense ribosome constructs containing 100-bp DNA segments derived from the 5′-ends, 3′-ends, and internal coding regions of two non-essential genes, granule lattice protein 1 and macronuclear histone H1. The 5′- and 3′-end constructs inhibited gene expression, but antisense ribosomes derived exclusively from coding regions had little effect.