Loss of p16 expression can occur via homozygous deletion, point mutation, or hypermethylation of exon 1. Astrocytomas representing all World Health Organization (WHO) grades of malignancy were analyzed in a correlative study using multiplex polymerase chain reaction (PCR) analysis to detect deletions of the p16 gene together with immunohistochemistry to detect loss of the protein in archival specimens of the same tumors. Homozygous deletions of p16 were detected in 29% (15 of 52) of WHO grade 3 and 4 tumors. Immunostaining for p16 protein was present in 26 tumors retaining the p16 gene and absent in 11 tumors with deletions of the p16 gene. A close correlation was found between the two detection methods, with all tumors lacking immunostaining showing homozygous loss of the p16 gene. Astrocytomas exhibiting inactivation of the p16 gene most often contained p53 gene mutations or amplified epidermal growth factor receptor genes, genetic characteristics associated with both the progressive and de novo tumor variants. Immunohistochemical evaluation may be a useful, rapid method to screen astrocytomas for loss of p16 gene expression, regardless of the underlying mechanism leading to p16 gene inactivation.