Abstract: Retrotransposition of the Ty1 element of Saccharomyces cerevisiae is temperature sensitive. Transposition activity of Ty1 is abolished at temperatures above 34 degrees C. In this report, we show that the major block to transposition at high temperature is the inhibition of processing of the Gag-Pol-p199 polyprotein and the concomitant reduction of reverse transcriptase (RT) activity. Expression of a Ty1 protease construct in Escherichia coli shows that protease enzymatic activity is inherently temperature sensitive. In yeast, Gag processing is only partially inhibited at high temperature, while cleavage of the Gag-Pol polyprotein is completely inhibited. Sites of proteolytic processing are differentially susceptible to cleavage during growth at high temperature. Overall levels of the Gag-Pol polyprotein are reduced at high temperature, although the efficiency of the requisite +1 frameshifting event appears to be increased. RT activity is inherently relatively temperature resistant, yet no cDNA is made at high temperature and the amount of RT activity is greatly reduced in virus-like particles formed at high temperature. Taken together, these results suggest that alterations in Ty1 proteins that occur at high temperature affect both protease activity and RT activity, such that Ty1 transposition is abolished.