To further evaluate the effectiveness of the AutoCyte PREP thin-layer slide preparation (TriPath Imaging, Inc., Burlington, North Carolina) as compared to conventional Pap smears. A split-sample, blinded evaluation of matched thin-layer preparations and conventional smears from 2,438 patients was performed. This material was enriched by including 260 cases of high grade squamous intraepithelial lesions (HSILs) and cancer cases from an earlier study. Many of these cases were difficult to diagnose, containing very few abnormal cells on one or both matching slides. The preparations were evaluated multiple times by both thin-layer-inexperienced and -experienced cytology professionals to better compare performance related to preparation quality alone. The initial evaluations of the slides by personnel with only brief training in thin-layer interpretation demonstrated equivalent performance for the two preparations. The reevaluation study by cytology professionals with several months of thin-layer experience demonstrated a statistically significant improvement in detection of both LSIL and HSIL lesions using AutoCyte PREP slides. There was also a statistically significant improvement in the number of satisfactory samples using the AutoCyte PREP method. The study demonstrated that the AutoCyte PREP thin-layer slide preparation is at least equivalent to conventional Pap smears in the detection of LSIL and HSIL, even when evaluated by cytology professionals who have been newly trained in the thin-layer method and that, with increased experience, the thin-layer AutoCyte PREP slide preparation method showed a statistically significant improvement in disease detection.