Laser excitation wavelengths for two-line planar laser-induced fluorescence (PLIF) of 3-pentanone have been optimized for simultaneous imaging of temperature and composition under engine-relevant conditions. Validation of the diagnostic was performed in a motored optical IC engine seeded homogeneously with 3-pentanone. PLIF measurements of the uniform mixture during the compression stroke were used to measure the average temperature and to access the random uncertainty in the measurements. To determine the accuracy of the temperature measurements, experimental average temperatures were compared to values computed assuming isentropic compression and to the output of a tuned 1-D engine simulation. The comparison indicated that the absolute accuracy of the temperature measurements is better than ±5%. Probability density functions (PDFs) calculated from the single-shot images were used to estimate the precision of the measurements. The PDFs determined from the images had standard deviations as low as ±12 K for the temperature measurements. The diagnostic was demonstrated under conditions with controlled amounts of stratification induced by injection of cold unseeded fluid into the hot intake stream. Finally, the diagnostic was successfully tested during fired HCCI operation and was used to investigate the mixing of hot exhaust gas residuals with fresh intake charge.