The B subunit of Escherichia coli heat-labile toxin (LTB) is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. We previously examined high expression levels of LTB in plants by chloroplast and synthetic LTB gene expression and found substantially higher expression levels of LTB, compared to nuclear LTB expression in wild-type plants. The 2.5% LTB protein of total soluble protein that was observed by chloroplast transformation was approximately 250-fold greater expression than that of LTB via nuclear genome integration. In addition, the amount of LTB protein found in transgenic tobacco leaves using a synthetic LTB gene was 2.2% of the total soluble plant protein, which was approximately 200-fold higher than that in plants with native LTB gene expression. The purpose of our experiment was to increase LTB levels in plants by crossing chloroplast-transformed and synthetic LTB transgenic lines produced previously to express higher LTB levels. LTB protein levels in the F-1 transgenic tobacco plants was significantly higher (3.3%), compared to the 2.2% of chloroplast-transformed line or 2.8% of synthetic LTB gene line. Our results suggest that LTB expression was successfully enhanced in the F-1 hybrid generation of transgenic tobacco plants.