STAF [Sec (selenocysteine) tRNA gene transcription activating factor] is a transcription activating factor for a number of RNA Pol III- and RNA Pol II-dependent genes including the Trsp [Sec tRNA gene], which in turn controls the expression of all selenoproteins. Here, the role of STAF in regulating expression of Sec tRNA and selenoproteins was examined. We generated transgenic mice expressing the Trsp transgene lacking the STAF-binding site and made these mice dependent on the transgene for survival by removing the wild-type Trsp. The level of Sec tRNA was unaffected or slightly elevated in heart and testis, but reduced approximately 60% in liver and kidney, approximately 70% in lung and spleen and approximately 80% in brain and muscle compared with the corresponding organs in control mice. Moreover, the ratio of the two isoforms of Sec tRNA that differ by methylation at position 34 (Um34) was altered significantly, and the Um34-containing form was substantially reduced in all tissues examined. Selenoprotein expression in these animals was most affected in tissues in which the Sec tRNA levels were most severely reduced. Importantly, mice had a neurological phenotype strikingly similar to that of mice in which the selenoprotein P gene had been removed and their life span was substantially reduced. The results indicate that STAF influences selenoprotein expression by enhancing Trsp synthesis in an organ-specific manner and by controlling Sec tRNA modification in each tissue examined.