The VpreB/lambda5 surrogate L chain complex is an essential component of the pre-B cell receptor, the expression of which serves as an important checkpoint in B cell development. Surrogate L chains also may serve as components of murine pro-B cell receptors whose function is unknown. We have produced two new mAbs, R3 and R5, that recognize a different VpreB epitope than the one recognized by the previously described VP245 anti-mouse VpreB Ab. These Abs were used to confirm the expression of surrogate L chains on wild-type pro-B and pre-B cell lines. Although undetectable on the cell surface, VpreB was found to be normally expressed within B lineage cells of lambda5-deficient mice. Nevertheless, VpreB expression was extinguished at the B cell stage of differentiation in these mice. The normal pattern of VpreB expression in lambda5-deficient mice excludes an essential role for pro-B and pre-B cell receptors in VpreB regulation.