Genomic alteration near or within mitochondrial gene is often associated with cytoplasmic male sterility (CMS). Its influence on the expression of the mitochondrial gene was proposed as one of the possible causes of CMS. In sugar beet mitochondrial rps3, whose downstream 1,056-bp region contains Norf246, an apparently non-functional open reading frame (ORF), was deleted in CMS mitochondria. In our previous study, normal rps3 (3.8 kb), CMS rps3 (2.7 kb), and Norf246 (3.8 and 0.9 kb) were shown to be transcribed. The present study was conducted to determine whether the deletion affected gene expression. Reverse transcription (RT)-PCR analysis revealed the co-transcription of rps3 and Norf246. By circularized RNA (CR) RT-PCR analysis, the 5' and 3' termini of the 3.8- and the 0.9-kb transcripts were determined. The results suggested that the 3.8-kb transcripts were the rps3 mRNA bearing ~464-base 5' untranslated region (UTR) and ~1,508-base 3' UTR, whereas no functional ORF was observed in the 0.9-kb transcripts. CR-RT-PCR revealed that the 3' UTR of the 2.7-kb transcripts was reduced to ~460 bases. However, no difference in the accumulation of RPS3 polypeptide and RNA editing was detected by protein gel blot analysis and cDNA sequencing. Although the deleted region encoded the truncated-atp9 that was edited, no influence on the pattern and frequency of RNA editing of genuine atp9 was evident. The results eliminated rps3 as a candidate for the CMS gene, making preSatp6, a unique ORF fused with CMS atp6, the sole CMS-associated region in sugar beet.