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A single cylinder research engine with negative valve overlap (NVO) and direct gasoline injection was run in a homogeneous charge compression ignition (HCCI) mode. The split fuel injection technique was used, where the first injection was applied during exhaust re-compression, while the second injection was applied at the beginning of main compression. The quantities of the fuel injected at the two timings were varied from the whole fuel injection during NVO to the whole fuel injection during the main compression event. These split fuel ratio sweeps were repeated both for a stoichiometric mixture and for a slightly lean mixture. In the study, NVO reactions were assessed via analysis of the exhaust-fuel mixture composition after the NVO period and referred to the main event combustion. The results showed that fuel injection during NVO resulted in the production of substantial quantities of auto-ignition promoting species, such as acetylene and formaldehyde.