The present study characterized a mutation in the Eck receptor tyrosine kinase gene induced by the U3betageo gene trap retrovirus. The mutation (eck(i)) was identified during an in vitro screen for proviruses that disrupt developmentally regulated genes in cultured ES cells. The germ-line eck(i) fusion gene was expressed in blastocyst and later restricted to the primitive streak, node and to regions of the hindbrain in 6.5-10.5 day embryos. This is identical to the pattern of Eck gene expression as determined by either in situ hybridization or immunostaining, suggesting that expression of the Eck promoter was not affected by provirus integration. The provirus inserted approximately 8 kb upstream of the 5' end of the published cDNA sequence, and 1.8 kb downstream of an alternatively spliced 5' exon. The eck(i) allele is essentially a null mutation since mutant mice are severely deficient for Eck protein as determined by Western blot analysis and in vitro kinase assays. Nevertheless, mice homozygous for the mutation did not exhibit any discernable phenotype. These results suggest that other members of the Eph family of receptor tyrosine kinases can functionally compensate for loss of Eck.