We previously reported that passive transfer of polyclonal neutralizing antibodies (NAbs) sufficient to generate a titer of 1:38 in the plasma would confer sterilizing protection to 99% of macaques challenged intravenously with 75 TCID(50) of SHIV(DH12). Neutralizing activity in that study was measured in an MT4 cell assay in which infection was completely blocked (EC(100)). In the current study, the TZM-bl system was used to measure EC(50) neutralizing titers in several of the same macaque plasma samples and the relationship between these titers and in vivo protection was determined. The antiviral EC(50) NAb titers measured in individual plasma samples were higher than those previously obtained in the MT4 system. Furthermore, the geometric mean EC(50) NAb titers against pseudotyped SHIV(DH12) were 33-fold greater than the EC(100) titers measured in the MT4 cell assay against the replication-competent SHIV(DH12) inoculated into animals. An augmented probit regression model was used to generate curves relating TZM-bl EC(50) NAb titers and protection from a virus challenge; estimated titers conferring various levels of protection were then determined. In TZM-bl assays using pseudotyped SHIV(DH12), representative percent in vivo protection/estimated EC(50) titers were 99%/1:4467, 90%/1:1175, 80%/1:676, 50%/1:234, and 33%/1:141. Because it is likely that contributions from other arms of the immune system will contribute to vaccine-induced control, the range of EC(50) NAb titers we have derived may be more informative for evaluating the protective value of NAb activity from TZM-bl assays.