Random systematic mutagenesis was used to generate a library of human rhinovirus 14 chimeras that each display a segment from the V3 loop of human immunodeficiency virus type 1. The sequence XXIGPGRAXX, where X could be any of the 20 amino acids, was inserted at the neutralizing immunogenic site II of human rhinovirus 14 between VP2 residues 159 and 160. Twenty-five unique chimeric viruses were isolated, and the identity of their randomized residues was determined. A nonrandom amino acid distribution that may reflect structural requirements for viability was observed at the randomized positions. Fifteen of 25 chimeras were neutralized by one or more of a panel of four anti-human immunodeficiency virus type 1 V3 loop antibody preparations, indicating that antigenicity had been successfully transplanted. Libraries of chimeric viruses produced by using the techniques described may be a source of vaccines and other immunotherapeutic reagents. The random systematic mutagenesis methodology described should be generally useful for the rapid transplantation of foreign sequences into viral coat and other proteins to produce libraries containing members with the desired properties.