The objective of the present study was to assess the effect of tobacco chewing on buccal mucosa by using cytomorphometry. We compared the cellular diameter (CD), nuclear diameter (ND) and the ratio of nuclear diameter to cellular diameter (N/C) of buccal mucosa squames of normal subjects (N) with buccal mucosa squames of tobacco users without lesion (A), with tobacco-lime lesion (B), leukoplakia (C), and oral squamous cell carcinoma (D). The study group consisted of 125 patients divided into five groups (N, A, B, C and D) between the ages of 21 and 75 years. The mean of the cellular diameter (in micrometers) of group N, A, B, C, and D was 72.86+/-5.26, 68.30+/-3.02, 62.13+/-3.29, 57.75+/-4.66, 54.51+/-4.66 respectively (p<0.01). The mean of the nuclear diameter (in micrometers) of group N, A, B, C, and D was 8.70+/-1.30, 8.98+/-1.08, 9.06+/-0.83, 9.12+/-1.06, and 11.04+/-1.46 respectively (p<0.01). The mean of the ratio of nuclear diameter to cellular diameter of group N, A, B, C, and D was 0.11+/-2.00, 0.13+/-1.82, 0.14+/-1.35, 0.16+/-3.11, 0.21+/-4.51 respectively (p<0.01). Univariate analysis of variance (ANOVA) showed a significant group effect for cellular diameter, nuclear diameter and ratio of nuclear diameter to cellular diameter. Multiple comparison tests by Tukey-HSD procedure revealed a significant decrease in the mean cellular diameter, increase in the nuclear diameter and ratio of nuclear diameter to cellular diameter. Cytomorphometric changes could be the earliest indicators of cellular alterations. There is progressive decrease in cellular diameter, increase in nuclear diameter and increase in ratio of nuclear diameter to cellular diameter in smears from all tobacco users, as compared to normal subjects. This indicates that there could be cause-effect relationship between tobacco and quantitative alterations.