Natural killer cells are important in innate defense against viral infections. The interplay between stimulatory and inhibitory natural killer cell receptors and their corresponding human leukocyte antigen ligands are known to influence the outcome of acute Hepatitis C virus infection. Frequencies of NK receptor genes (8 inhibitory, 6 activating and 2 pseudogenes) and HLA class II alleles (DRB1, DQB1) were analyzed in 160 Puerto-Rican American drug users with Hepatitis C virus infection; 121 had chronic viremia (CV) and 39 were spontaneous clearance (SC). We further ruled out genetic stratification using short tandem repeats. Interaction between KIR gene receptor 2DL3/2DL3 and its ligand, C1/C1 of HLA-Cw alleles and spontaneous clearance was confirmed (p=0.03, OR=3.05). We also found a new interaction between the KIR receptor gene 2DL3 with HLA-DRB1*1201 (p=0.0001, OR=22) associated with SC, and an association of HLA DQB1*0501 (p=0.05, OR=0.30) with CV. Our findings suggested a role for MHC class II alleles in Hepatitis C virus peptide presentation to T cells together with NK ligand interaction involving pathways that will be useful for the development of immunotherapeutic interventions.