The purpose of the work was to determine the feasibility and predictive value of Ki-67 immunostaining of cervical cytology and the detection of cervical dysplasia. Air-dried cervical smears were stained with MIB-1 antibody to identify the Ki-67 antigen. Nuclear decoration in abnormal squamous nuclei determined immunoreactivity. One hundred twenty-four nonpregnant patients underwent colposcopy and directed biopsies for abnormal cytology. Sensitivity (0.89), specificity (0.65), positive predictive value (0.60), and negative predictive value (0.91) were found for Ki-67 immunostaining in detection of high-grade cervical intraepithelial neoplasia (CIN) in 124 patients and positive Ki-67 staining was a significant predictor of high-grade CIN in both univariate (odds ratio 15.5 (95% CI 5.5-43.8) and multivariable (odds ratio 21.5 (95% CI 5.0-92.0) analysis. In 101 patients with ASCUS and LGSIL, Ki-67 immunostaining demonstrated the following in detection of high-grade CIN: sensitivity (0.96), specificity (0.67), positive predictive value (0.49), and negative predictive value (0.98). Ki-67 immunostaining of cervical cytology is a predictor of significant cervical pathology with high sensitivity and negative predictive value. Ki-67 immunostaining of cervical cytology may represent a new and cost-effective triage tool for patients with minor abnormalities on cytology.