We have shown previously that a significant number of invasive cervical cancers (ICC) have nonrandom chromosomal losses in 3p, 6p, 11q, 2q, 6q, and 19q, thereby suggesting that genes involved in the suppression of tumor development or progression are located in these regions. Cervical intraepithelial neoplasia (CIN) III is considered the precursor lesion for ICC of squamous type and occurs frequently with ICC of glandular type. In an effort to define which chromosomal losses are present in the precursor lesions, we identified CIN III lesions from 24 ICC treated by radical hysterectomy. Thirty-three CIN III associated with 22 squamous carcinomas and 2 adenocarcinomas were carefully microdissected from the paraffin-embedded sections. The whole genomic DNA from CIN III was amplified with short random primers. DNA from ICC, CIN III, and normal tissue was analyzed at the six chromosomal regions with polymorphic markers. Thirty-eight percent of hysterectomy specimens had loss of heterozygosity (LOH) in at least one of the CIN III lesions from each case. Loss occurred in 30% of cases in 3p14.1-12 (37% for associated ICC), 21% in 6p23 (33%), 14% in 2q33-37 (27%), 0 in 11q23.3 (33%), 4% in 19q13.4 (13%), and 0 in 6q21-23.3 (18%). These results suggest that mutations in 3p and 6p are important early in tumorigenesis, whereas 11q and 6q contain genes important later in tumor progression. Invasive and preinvasive cervical lesions appear to develop from multifocal genetic events since consistent losses do not occur within all precursor lesions in the same patient.