The protein tyrosine kinase, p56lck, is involved in signal transduction in mature T cells and in the molecular events controlling early thymocyte differentiation. Thymuses of mice deficient for p56lck expression (p56lck-/-) consist of immature CD4-CD8- double-negative (DN) and CD4+CD8+ double-positive (DP) thymocytes and are severely reduced in total cell number. In this report we have studied DN thymocytes from p56lck-/- mice and found an increase in the proportion of the CD44-CD25+ subset, suggesting that transit through this stage, which is known to require T cell receptor (TcR) beta expression, may be delayed in the absence of p56lck expression. In addition, the expression of a transgenic TcR beta chain or TcR alpha beta pair did not restore thymic development in p56lck-/- mice. However, in contrast to mice expressing a dominant negative isoform of p56lck in which DP thymocytes do not develop, DP thymocytes still develop in nontransgenic and TcR transgenic p56lck-/- mice. These results demonstrate that expansion of the DP subset is impaired in p56lck-/- mice. In contrast, allelic exclusion is not severely compromised. Although there was an increase in the number of peripheral T cells expressing more than one V beta chain in TcR transgenic p56lck-/- mice, we found that inhibition of endogenous TcR beta gene rearrangement was almost complete in thymocytes of V beta transgenic p56lck-/- mice and we could not detect any peripheral T cells that expressed more than one V beta chain in non-transgenic p56lck-/- mice.