The primary aims of this study were to establish a high-throughput assay and analytic procedures that would allow us to assess with accuracy and reproducibility the presence of anti-cyclin B1 antibodies in a large cohort of subjects at-risk for development of lung cancer. The overall goal is to identify factors significantly associated with immune responses against this lung tumor antigen and to evaluate the relationship between anticyclin B1 antibodies and lung cancer risk in a prospective cohort. Successful cancer treatment relies on early detection. The immune system could respond to tumor antigens arising from altered host factors and provide an early readout for cancer presence. This study examines immune responses to the tumor antigen cyclin B1. Lung cancer patients have antibodies against cyclin B1. Unscheduled overexpression of this cell cycle regulator occurs early in tumorigenesis and might be detected by the immune system long before clinical diagnosis of cancer. Antibodies recognizing cyclin B1 were measured using semiautomated ELISAs in sera samples from subjects without detectable lung cancer enrolled in the Pittsburgh Lung Screening Study (PLuSS), a longitudinal study of long-term smokers over age 50. Factors significantly associated with antibody presence were identified using linear regression analysis. We have established a highly reproducible, semiautomated ELISA-based assay for the high-throughput assessment of serum antibody titers. Using this technology and a small subset of PluSS subjects, cyclin B1 antibody levels were found to be high in a small proportion of subjects tested. Regression analysis identified gender as well as age in women smokers to be significant determinants of cyclin B1 antibody levels, association not seen in male subjects. This work represents an important first step toward defining the importance of the presence of anticyclin B1 antibodies in an at-risk population and assessing the predictive value of serum cyclin B1 antibody as relates to lung cancer risk.