It has been known that factor XIa (XIa) can react covalently with antithrombin III (ATIII) to form two complexes with ratios of 2ATIII:1XIa and 1ATIII:1XIa. In the hands of these investigators, the M.W.s, as measured by SDS-PAGE, were 265 kDa and 225 kDa, respectively. In this investigation it has been observed that the addition of 1 μg or 5 μg heparin (H) to 3.1 × 10-5 (mol ATIII for 5 minutes prior to the addition of 1.125 μ 10-5 μmol XIa for a 30-minute incubation led to an increase in the 265 kDa band of 113% and 223%, respectively. These results were statistically significant (p < .01). However, when H was premixed with XIa first, before the addition of AT III, statistical increases in the 265 kDa band were also seen (267% and 183%, respectively; p < .0005, n = 6). Protamine sulfate (PS) statistically significantly (p < .05) inhibited the formation of the 265 and 225 kDa XIa-ATIII complexes at the 5 μg PS level when premixed with ATIII or XIa, respectively, the decreases in the 265 kDa band being 39.1 and 34.4%, respectively for [(ATIII/PS) + XIA] and [(XIa/PS) + ATIII] mixtures, and 23.1 and 23.8% for the 225 kDa band with [(ATIII/PS) + XIa] and [(XIa/PS) + ATIII] mixtures. These results with PS indicate that PS inhibits complex formation between XIa and ATIII at the 2ATIII:1 XIa and 1ATIII:1 XIa levels, in contrast with its reported stimulation of complex formation between thrombin and ATIII.