Ribonuclease P (RNase P) is involved in regulation of noncoding RNA (ncRNA) expression in Saccharomyces cerevisiae. A hidden-in-reading-frame antisense-1 (HRA1) RNA in S. cerevisiae, which belongs to a class of ncRNAs located in the antisense strand to verified protein coding regions, was cloned for further use in RNase P assays. Escherichia coli RNase P assays in vitro of HRA1 RNA show two cleavage sites, one major and one minor in terms of rates. The same result was observed with a partially purified S. cerevisiae RNase P activity, both at 30 degrees C and 37 degrees C. These latter cells are normally grown at 30 degrees C. Predictions of the secondary structure of HRA1 RNA in silico show the cleavage sites are canonical RNase P recognition sites. A relatively small amount of endogenous HRA1 RNA was identified by RT-PCR in yeast cells. The endogenous HRA1 RNA is increased in amount in strains that are deficient in RNase P activity. A deletion of 10 nucleotides in the HRA1 gene that does not overlap with the gene coding for a protein (DRS2) in the sense strand shows no defective growth in galactose or glucose. These data indicate that HRA1 RNA is a substrate for RNase P and does not appear as a direct consequence of separate regulatory effects of the enzyme on ncRNAs.