Despite a simple consensus sequence, there is considerable variation of promoter strengths, transcription rates, and the kinetics of initiating nucleotide incorporation among the promoters found in the Saccharomyces cerevisiae mitochondrial genome. We asked how changes in the initiating (+1 and +2) nucleotides, conformation of the promoter DNA template, and mutation of the mitochondrial RNA polymerase (mtRNAP) affect the kinetics of nucleotide (NTP) utilization. Using a highly purified in vitro mitochondrial transcription system, we found that 1) the mtRNAP requires the highest concentrations of the +1 and +2 initiating NTPs, intermediate concentrations of NTPs at positions 5 to 11, and low concentrations of elongating NTPs; 2) the mtRNAP requires a higher concentration of the +2 NTP than the +1 NTP for initiation; 3) the kinetics of +2 NTP utilization are altered by a point mutation in the mtRNAP subunit Mtf1; and 4) a supercoiled or pre-melted promoter DNA template restores normal +2 NTP utilization by the Mtf1 mutant. Based on comparisons to the structural and biochemical properties of the bacterial RNAP and the closely related T7 RNAP, we propose that initiating nucleotides, particularly the +2 NTP, are required at high concentrations to drive mitochondrial promoter opening or to stabilize a productive open complex.