Most of the previous research on flame ionization in spark ignition engines applied positive polarity on the spark plug center electrode, referred to as positively biased probe. In this paper an investigation is made to determine the characteristics of the ion current signal with negatively biased probe. The factors that contribute to the second ion current peak, reported to be missing with negative polarity, are investigated. Experiments were conducted on a research single-cylinder, spark ignition engine and the negative polarity is applied by a SmartFire Plasma Ignition system. The effect of different spark plug designs and engine operating parameters on the amplitude and timing of each of the two ion current peaks is determined. The results indicated that, with negative polarity, the cathode area is one of the main factors that contribute to the amplitude of the ion current signal, particularly the second peak.