Human papillomavirus (HPV) infection has been implicated as an etiologic factor in most cervical cancers. However, additional genetic alterations are thought to be required for the development of a carcinogenic genotype. In the present study, interphase cytogenetics utilizing pericentromeric probes specific for chromosomes 1, 3, 11, 17, 18, and X was performed on paraffin-embedded tissue sections from 25 high-grade squamous intraepithelial lesions (SILs) and 25 invasive squamous cell carcinomas (ISCCs) of the cervix. HPV infection was determined by both in situ hybridization and broad-spectrum GP5+/GP6+ PCR. HPV was identified in all high-grade SILs (HPV 16, n = 16; 18, n = 2; 26, n = 1; 31, n = 4; 45, n = 1; 66, n = 1) and 23 (92%) ISCCs (HPV 16, n = 19; 18, n = 2; 31, n = 1; 39, n = 1). Aneusomy was identified in 11 (44%) high-grade SILs and 18 (72%) ISCCs. In 18 (62%) of these, relative under-representation of chromosomes 3, 11, 17, and/or 18 was identified (8 high-grade SILs and 10 ISCCs). Tetrasomy of all six chromosomes was present in two high-grade SILs but no ISCCs. Twelve (48%) high-grade SILs and seven (28%) ISCCs were disomic with all six chromosome probes, and there was no relationship between HPV presence or type and chromosome pattern. The presence of distinct patterns of numerical chromosome abnormality in these lesions suggests that progression to high-grade SIL or invasive carcinoma can occur by more than one genetic pathway. The lack of correlation between chromosome pattern and HPV type indicates that these pathways are not HPV type-specific. Whether these patterns reflect differences in early gene expression, possibly related to viral integration, or differences in the biologic properties of HPV type variants remains to be established.