The genes responsible for the development of neuroblastoma following in vivo deletion or mutation are largely unknown. We have performed loss of heterozygosity studies on a series of 24 Portuguese primary neuroblastomas using 6 polymorphic markers located at chromosome 9p21 spanning the p16/MTS1/CDKN2, p15/MTS2/CDKN2B, and the interferon alpha and beta genes. Loss of heterozygosity was observed in 4 of the 24 tumors (17%), a somewhat lower percentage than a previous study that identified patients by a mass screening program. A correlation was also observed between 9p21 LOH and 1p36 LOH in our group of tumors. Two distinct regions of 9p21 deletion were observed: one located in the region adjacent to the markers D9S162 and D9S1747 and a second located centromerically of the p16 gene near the D9S171 marker. The latter region is exclusive of the p16 gene. This result suggests the presence of at least one other tumor suppressor gene at 9p21, apart from the p16 and p15 genes, which may be of importance to the development of neuroblastoma.