Cervical cancer is a malignancy closely associated with human papillomavirus (HPV). However, some cervical carcinomas occur in the absence of the virus. Two HPV-negative cervical carcinoma cell lines, C-33A and HT-3, were examined by G-banding and fluorescence in situ hybridization (FISH) using several whole chromosome and satellite probes. Combined conventional banding and FISH analysis significantly enhanced identification of complex and cryptic chromosome rearrangements. Common to both cell lines were translocations of chromosome 1, involving a specific site on the short arm and partial or complete loss of the short arm of chromosome 9, as well as loss of chromosome 13. The C-33A line had a relatively simple chromosomal constitution, with chromosome 1 being involved in translocations with chromosomes 9, 18, and 21. Karyotype reconstruction of G-banded and painted chromosomes indicate a net loss of DNA as a result of unbalanced translocations, which occurred only at site 9p24 and loss of one copy of chromosomes 13 and 14. In contrast, HT-3 cells had complex rearrangements and deletions of chromosomes 1p, 3p, 9p, 10q/p, 11p/q, and 17p, all regions with known tumor suppressor genes. The deletions observed in these HPV-negative cervical carcinomas will be important in delineating regions of tumor suppressor genes.