Certain VH genes are predominantly expressed in mature B cells. We hypothesized that several, mutually nonexclusive VH-dependent mechanisms operating at distinct stages during B cell development may be responsible for overrepresentation of these VH genes. In the present study, we have assessed whether one of the mechanisms involves preferential rearrangement at the pro-B cell stage. The frequency of individual VH4 and VH3 genes in rearrangement libraries from FACS-purified human CD34+/CD19+ pro-B and CD34-/CD19+ pre-B cells was assessed. The in-frame and out-of-frame rearrangements from both cell populations were analyzed using a high resolution PAGE system. The frequencies of individual VH gene segments among out-of-frame rearrangements from pro-B cells were determined, because these frequencies should reflect only processes before the translation of the mu-heavy chain and should not be biased by selection mechanisms. Our results demonstrate that, at the pro-B cell stage, the V4-34, V4-39, and V4-59 gene segments are the most frequently rearranged VH4 family genes, and the V3-23 and V3-30 gene segments are the most frequently rearranged VH3 family genes. This finding suggests that the predominant expression of these VH genes in peripheral mature B cells is determined to a significant degree by their preferential rearrangement during V-DJ recombination.