Background: Acquisition of human immunodeficiency virus type 1 (HIV-1) infection is mediated by a combination of characteristics of the infectious and the susceptible member of a transmission pair, including human behavioral and genetic factors, as well as viral fitness and tropism. Here we report on the impact of established and potential new HLA class I determinants of heterosexual HIV-1 acquisition in the HIV-1-exposed seronegative (HESN) partners of serodiscordant Zambian couples. Methodology/Principal Findings: We assessed the relationships of behavioral and clinically documented risk factors, index partner viral load, and host genetic markers to HIV-1 transmission among 568 cohabiting couples followed for at least nine months. We genotyped subjects for three classical HLA class I genes known to influence immune control of HIV-1 infection. From 1995 to December 2006, 240 HESNs seroconverted and 328 remained seronegative. In Cox proportional hazards models, HLA-A*68:02 and the B*42-C*17 haplotype in HESN partners were significantly and independently associated with faster HIV-1 acquisition (relative hazards = 1.57 and 1.55; p = 0.007 and 0.013, respectively) after controlling for other previously established contributing factors in the index partner (viral load and specific class I alleles), in the HESN partner (age, gender), or in the couple (behavioral and clinical risk score). Few if any previously implicated class I markers were associated here with the rate of acquiring infection. Conclusions/Significance: A few HLA class I markers showed modest effects on acquisition of HIV-1 subtype C infection in HESN partners of discordant Zambian couples. However, the striking disparity between those few markers and the more numerous, different markers found to determine HIV-1 disease course makes it highly unlikely that, whatever the influence of class I variation on the rate of infection, the mechanism mediating that phenomenon is identical to that involved in disease control. © 2011 Song et al.