Objective: Previously we have shown that a malignant mouse keratinocyte cell line, 10Gy5, has elevated AP-1 transactivation and reduced JunB protein levels compared to its parental benign cell line, 308, and that the tumorigenicity in the 10Gy5 cells could be blocked by a dominant negative c-Jun mutant protein. We wished to determine whether the change in JunB protein levels could account for the elevated AP-1 activity and whether re-expression of JunB in malignant 10Gy5 cells altered their proliferative capacity. Design: In the current study, we reduced JunB expression in benign 308 cells with antisense oligonucleotides and increased JunB expression in malignant 10Gy5 cells by stable transfection of a JunB expression vector. Results: Increased AP-1 activity was detected after treatment of the benign 308 cell line with JunB antisense oligonucleotides that reduced JunB protein levels. Stably JunB-transfected clones of malignant 10Gy5 cells showed decreased AP-1 activity, slowed in vitro cell proliferation and reduced tumor growth when xenografted to athymic nude mice. Conclusion: These findings suggest that expression of JunB protein has a negative effect on malignant tumor cell proliferation in part through its ability to inhibit AP-1 transactivation.