We examined the genomic status of the p16 gene in 5 human glioma cell lines by Southern blot analysis. The p16 gene was located in the 9p21 chromosomal region and homozygous deletion was detected in 4 of 5 (80%) human glioma cell lines and 5 of 15 (33%) clinical samples. We transfected the full-length human p16 gene into p16-null human glioma cell line, U251MG cells, using the plasmid vector pRc/CMV-p16 and evaluated the effect of p16 gene transfer on the growth suppression of malignant glioma cells. The transfection of p16 cDNA caused growth suppression through G1 cell cycle arrest in U251MG cells. We also examined the effect of p16 gene transfer on the chemosensitivity to cis-diamminedichloroplatinum II (CDDP), 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl) -3-nitrosourea hydrochloride (ACNU), and 5'-azacytidine (AZC). We did not detect any change in them after p16 gene transfer. These results might suggest that deletion of p16 genes promoted unrestrained growth in human glioma but has no relationship to the chemosensitivity to CDDP, ACNU and AZC.