SHP-1 protein tyrosine phosphatase is a critical regulator of signal transduction in hematopoietic cells. In the present study, we derived two pre-B cell lines, PBCL-1 and PBCL-2, from normal and SHP-1-deficient motheaten mice, respectively, and characterized hyperphosphorylated proteins in PBCL-2 cells to identify SHP-1-regulated molecules. Two proteins of 56 and 53 kDa (p56/p53) in PBCL-2 cells showed heightened phosphorylation (3- to 6-fold) in comparison with those in PBCL-1. p56/p53 were identified as the two forms of the lyn protein tyrosine kinase (p56/p53lyn), which showed increased kinase activity in PBCL-2 cells. Interestingly, the protein levels of p56/53lyn were found to be 3- to 6-fold higher in PBCL-2 cells than those in PBCL-1, whereas the transcript levels of lyn in the two cell lines were comparable. A modest increase in p56/53lyn protein expression was also detected in primary spleen B cells of motheaten mice. Thus SHP-1 deficiency in B-lineage cells, especially pre-B cells, is associated with increased lyn protein expression and kinase activity. These data indicate a role for SHP-1 in regulating lyn through a post-transcriptional mechanism.