p16 (CDKN2/MTS1/p16INK4a) is frequently deleted, methylated, or mutated in many malignancies including squamous cell carcinoma of the head and neck (HNSCC). p16 beta is an alternative transcript derived from a newly described exon (exon 1 beta) located more than 15 kb 5' to exon 1 of p16. Moreover, the p16 beta transcript theoretically encodes a protein distinct from p16 derived from a divergent reading frame putatively initiated in exon 1 beta. To test the contribution of both of these transcripts in carcinogenesis, full-length cDNA of p16 and p16 beta were cloned in separate vector constructs and then transfected into HNSCC cell lines characterized for p16 status (p16[+/+], p16[mut/-], and p16[methylated]). Transfection of either p16 or p16 beta resulted in marked growth inhibition in all three HNSCC lines tested, regardless of p16 status. However, p16 beta but not p16 inhibited the growth of HeLa cells, a cell line with inactive pRB due to expression of E7 papillomavirus protein. Moreover, transfection of all three HNSCC lines with either p16 or p16 beta resulted in a marked increase in cells in G0-G1 consistent with a cell cycle arrest in G1. These data are consistent with the hypothesis that p16 and p16 beta are growth-inhibitory genes active in HNSCC and that both act by blocking progression through the G1-S transition of the cell cycle. Furthermore, the suppressive effects of p16 beta on HeLa growth suggest that p16 beta mediates its effect independently from pRB.