LEDGF/p75 binding-defective IN mutant viruses were previously characterized as replication-defective, yet RNAi did not reveal an essential role for the host factor in HIV-1 replication. Correlative analyses of protein binding and viral fitness were expanded here by targeting 12 residues at the IN-LEDGF/p75 binding interface. Whereas many of the resultant viruses were defective, the majority of the INs displayed wild-type in vitro integration activities. Though an overall trend of parallel loss of LEDGF/p75 binding and HIV-1 infectivity was observed, a strict correlation was not. His-tagged IN(A128Q), derived from a phenotypically wild-type virus, failed to pull-down LEDGF/p75, but IN(A128Q) was effectively recovered in a reciprocal GST pull-down assay. Under these conditions, IN(H171A), also derived from a phenotypically wild-type virus, interacted less efficiently than a previously described interaction-defective mutant, IN(Q168A). Thus, the relative affinity of the in vitro IN-LEDGF/p75 interaction is not a universal predictor of IN mutant viral fitness.