High proliferative fraction and cyclin D1/CCND1 gene amplification have been associated with certain aggressive features of head and neck squamous carcinoma in some studies, but not in others. The differences may be related to the intratumoral heterogeneity of these factors. Moreover, the interrelationship between these seemingly related factors has not been determined. In order to determine the correlation between tumor proliferative fractions and CCND1 gene amplification, 3 spatially different samples from each of 32 primary head and neck squamous cell carcinomas (HNSCC) were separately analyzed by flow cytometry. A mixture of these specimens was also minced and snap frozen for molecular studies. DNA was extracted from patient lymphocytes, normal appearing squamous epithelium, and tumors. A genomic DNA probe containing the first exon of CCND1 was used for hybridization by Southern technique. A 5.6 kb genomic DNA probe of immunoglobulin heavy chain was used as an internal standard for quantification of CCND1 gene amplification. Our results showed that 30% of the tumor cases manifested intratumoral heterogeneity (> 50% differences) of their proliferative activity. The highest value was used for correlation with gene amplification. Eleven (34.4%) of the 32 tumors showed CCND1 amplification (2-10-fold). When the proliferative fraction was dichotomized into high and low groups based on the mean value (> or = 13%), a highly statistical correlation between CCND1 amplification and tumor proliferation was obtained (P < 0.001). No significant correlation between gene amplification and other clinicopathologic parameters was noted.(ABSTRACT TRUNCATED AT 250 WORDS)