The antiproliferative potential of hexamethylene diamine (HMDA) for mitogen-stimulated splenic lymphocytes was evaluated in vitro at final concentrations of 0.1-16 mM. Addition at the start of culture or after 24 or 48 h of culture decreased the proliferative response to T and B cell mitogens. However, the concentration of HMDA required to cause suppression increased with incubation time. Removal of diamine after 24 h allowed cells to proliferate normally upon reculture with mitogen. ODC activity, which was much greater in cultures stimulated with Con A than LPS, was markedly decreased by inclusion of diamine or DFMO in the culture medium. Addition of putrescine to cultures did not reverse the suppressive effects of diamine on proliferation but did restore DFMO-containing cultures to control levels of activity. These results indicate that HMDA does suppress lymphocyte proliferation in vitro by alteration of ODC and polyamine activity. However, comparison of results obtained with DFMO and HMDA suggests that HMDA may act via multiple pathways, only one of which involves inhibition of ODC activity. (Copyright (c) 1989 Elsevier Scientific Publishers Ireland Ltd.)