Mitochondrial RNA polymerase activity has been isolated from the crustacean Artemia franciscana at two stages of development, dormant embryo and developing larva. The preparations were obtained from purified mitochondria and the polymerase activity was purified by heparin-Sepharose chromatography. The presumed polymerase has a molecular mass of about 120 kDa and a 7.4 S sedimentation coefficient. The biochemical characterization of the enzymatic reaction identified our RNA polymerase preparations as mitochondrial. The transcription initiation sites of Artemia mtDNA were characterized recently in our laboratory (J. A. Carrodeguas and C. G. Vallejo, Eur. J. Biochem. 250, 514-523, 1997). Artemia mtDNA fragments comprising the transcription initiation sites were transcribed by the partially purified polymerase preparation from the two developmental stages, but the transcription turned out to be unspecific. DNAse I footprinting analysis of a main transcription initiation site-containing DNA fragment revealed a protected region around the initiation site +1 position, when using a crude polymerase preparation. However, the protected region was not observed with the purified preparation. The results altogether suggest that a specificity factor is lost during purification. Based on the footprinting data, we suggest that the sequence from positions -6 to +13 of the main transcription initiation site in the Artemia mitochondrial DNA is the binding site of the homologous RNA polymerase holoenzyme.