The nucleolar Mak16p protein of Saccharomyces cerevisiae has been implicated in 60S ribosome biogenesis. To learn more about the role of Mak16p in this process, ribosomal RNA processing was examined in a mak16-1 temperature-sensitive yeast strain. Steady-state levels of the 25S and 5.8S mature rRNA species dropped dramatically over a 4 h period in the mak16-1 yeast after a shift to the non-permissive temperature, while 18S and 5S rRNA levels decreased only moderately. Ribosomal RNA processing (rRNA) analyses showed that the most prominent defect at the non-permissive temperature was a dramatic decrease in 27SB precursor RNA levels, with no significant increase in the levels of any precursor. These data indicate an essential role for Mak16p in the stability of the 27SB precursor rRNA. Association of Mak16p with the 66S preribosomal complex does not appear to be sufficient for its function, because the mutant Mak16-1p protein was detected in sucrose density gradient fractions corresponding to the 66S pre-RNP complex.