It has been extremely difficult to elicit broadly cross-reactive neutralizing antibodies (Nabs) against human immunodeficiency virus type 1 (HIV-1). In this study, we compared the immunogenic properties of the wild-type and variable loop-deleted HIV-1 envelope glycoproteins. Mice were immunized with recombinant vaccinia viruses expressing either the wild-type or the variable loop-deleted (V1-2, V3, V4, and V1-3) HIV-1(DH12) gp160s. The animals were subsequently boosted with respective recombinant gp120s. All envelope constructs elicited similar levels of gp120-binding antibodies when analyzed by enzyme-linked immunosorbent assay (ELISA). However, the highest neutralizing activity was observed in sera from animals immunized with the wild-type envelope protein, followed by those immunized with DeltaV4 and DeltaV1-2. No neutralizing activity was detected in sera from animals immunized with DeltaV3 or DeltaV1-3. To identify immunogenic epitopes, ELISA was performed with overlapping 15-mer peptides that cover the entire length of gp120. For the wild-type gp120, the immunogenic epitopes mapped primarily to the variable loops V1-2 and to the conserved regions C1 and C5. When they were plotted onto known coordinates of gp120 core crystal structure, the epitopes in the conserved regions mapped predominantly to the inner domain of the protein. By immunizing with variable loop-deleted envelopes, the immune responses could be redirected to other regions of the protein. However, the newly targeted epitopes were neither on the exposed surface of the protein nor on the receptor binding regions. Interestingly, the removal of the V3 loop resulted in loss of immunoreactivity for both V3 and V1/V2 loops, suggesting structural interaction between the two regions. Our results suggest that obtaining broadly reactive Nabs may not be achieved simply by deleting the variable loops of gp120. However, the observation that the immune responses could be redirected by altering the protein composition might allow us to explore alternative strategies for modifying the antigenic properties of HIV-1 envelope glycoprotein.