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Objective: Evaluate peripheral blood lymphocyte proliferation (replicative index:RI) and micronuclei frequency (MF) among 2,4–D herbicide applicators. Methods: Twelve applicators spraying only 2,4–D provided a blood and urine specimen upon enrollment, several urine samples during the spraying season, and a blood specimen at the study's end. Nine controls provided blood and urine specimens upon enrollment and at the study's end. Gas chromatography/tandem mass spectroscopy determined urinary 2,4–D levels and standard in-vitroassays determined RI and MF scores. Applicator RI and MF were compared before and after spraying and with controls. Results: Applicators contributed 45 urine specimens with concentrations ranging from 1.0 to 1700 (μg 2,4–D/g creatinine/L urine) that logarithmically (ln) increased as spraying time increased. Applicator RI increased after spraying (p= 0.016), independent of tobacco and alcohol use, and demonstrated a weak dose-response with increasing urinary 2,4–D levels (p= 0.15). Among 2,4–D applicators, pre-exposure complete blood counts and lymphocyte immunophenotypes were not significantly different from post-exposure measurements. Conclusion: Urinary 2,4–D concentration, an exposure biomarker, may be associated with lymphocyte replicative index, a cell proliferation biomarker.