The gene that encodes the general transcription factor known as BTF3 was disrupted in mouse embryonic stem cells in a random mutagenesis screen for developmental genes with the ROSA beta-geo (Friedrich and Soriano, 1991) retroviral gene trap vector. The BTF3 mutation was transmitted through the germline of chimaeric mice. While the endogenous BTF3 gene is ubiquitously expressed, the expression pattern of the beta-galactosidase reporter gene present in the gene trap vector in BTF3 heterozygotes was restricted. Mice homozygous for the mutant allele died soon after implantation, around embryonic day 6.5. Thus, BTF3 is essential for postimplantation development. The isolation of the BTF3 sequences in this ROSA beta-geo insertion was facilitated by a relatively simple single lacZ primer reverse transcription PCR strategy.