We describe a procedure for quantification of mitochondrial (mt) RNA present in total RNA extracts of HT-29 human colonic adenocarcinoma cells grown under conditions for rapid growth (25 mM glucose) or differentiation (25 mM trehalose or 5 mM butyrate). Purified mt DNA was fragmented into specific coding regions using restriction endonuclease sites predicted from HeLa cell mt DNA sequence and probed with either 32P-labelled mt DNA or cDNA made from total RNA of HT-29 or HeLa cells. The amounts of probe that hybridized to various gene-encoded mt DNA fragments or RNA were quantified by laser densitometry. Use of 13 restriction endonucleases revealed that most if not all the mt DNA of HT-29 and K562 leukemic cells was comparable in size to that of HeLa cells. Relative levels of mt RNA from rapidly growing HT-29 and HeLa cells were lower than those measured for differentiated HT-29 cells induced by either trehalose or butyrate. In rapidly growing HT-29 cells and HeLa cells, the highest levels of specific mt RNAs were those encoded by mt DNA sequences immediately flanking the nested promoters and the heavy-strand replication origin (OH). Expression patterns of specific mt RNAs from HT-29 cells treated with butyrate and with trehalose were similar, but not identical. In either case, the mt RNAs that increased the most were those coded by mt DNA sequences located downstream from the light-strand replication origin (OL), suggesting a novel pattern of expression not seen before.