Few studies have reported background levels for nickel (Ni) in semen even though Ni is ubiquitous and humans are exposed. Fractionated semen (i.e., spermatozoa, seminal fluid) from ten men and a pooled sample (N=3) estimated the background level for Ni at 0.01 mug/mL using PIXE and ICP-MS. Ni(II) has been shown to bind a synthesized fragment of human protamine 2 (HP2), a protein involved in sperm chromatin compaction. HP2 binds one atom of zinc. High Performance Liquid Chromatography (HPLC), acid-urea polyacrylamide gel electrophoresis, and Western Blot analysis using the monoclonal antibody Hup2b served to purify and identify HP2 from mature differentiated spermatozoa; however, the yield was low (∼300--400mug). Low yield may have been a by-product of the quality or quantity of semen samples collected. Therefore, the purity of synthesized HP2 was confirmed prior to studying the interaction between Ni(II) and HP2. The absorption spectra of HP2 in the presence of an increasing number of molar equivalents of Ni(II) showed 2:1 binding. The stability constants were log beta1=7.7 and beta2=13.8 suggesting one weak site of Ni(II) coordination and one strong site of Ni(II) coordination to HP2. Incremental additions of Zn(II) to Ni(II)-HP2 resulted in the total loss of Ni(II) coordination at ∼335 nm suggesting Zn(II) either displaced one Ni(II) from HP2 (i.e., from the weak site) or altered the structure of HP2 so that one Ni(II) could no longer bind. The physiologically relevant condition, Zn(II)-HP2 also showed 2:1 binding upon addition of an increasing number of molar equivalents of Ni(II). The stability constants for Ni(II) binding Zn(II)-HP2 were log beta1=7.9 and log beta2=14.6 suggesting strong Ni(II) coordination to HP2 in the presence of Zn(II). Nickel has not been unequivocally established as a male reproductive toxin, but the interaction with HP2 in the presence of Zn(II) suggests exposure to Ni(II) during spermiogenesis may influence sperm chromatin remodeling and stability. It is important to study this interaction because altering sperm chromatin packaging and stability may lead to adverse male reproductive health effects, male factor infertility, or adverse pregnancy outcomes in the progeny of men occupationally exposed to nickel.