Each variant surface glycoprotein (Vsg) expression site (ES) in bloodstream-form Trypanosoma brucei is a polycistronic transcription unit containing several distinct expression site-associated genes (esag), in addition to a single vsg gene. esag1 genes from different ESs encode a highly polymorphic family of membrane-associated glycoproteins, whose function is unknown. In the hope of producing a phenotype that could indicate a function, we disrupted the esag1 genes in two ESs by targeted insertion of a hygromycin phosphotransferase gene. Our failure to produce an obvious phenotype prompted us to search for other esag1 transcripts. RNA from the mutant trypanosomes hybridized with an esag1-specific oligonucleotide. Cloning and sequencing of mRNA from both mutant and wild-type cells showed that several esag1 family members were expressed, each at a much lower level than the esag1 transcript from the active ES in wild-type trypanosomes. Long-range DNA mapping showed that these additional esag1 genes, some of which contained premature translation-termination codons, most probably originate from chromosomal-internal genes and pseudogenes. We have therefore been unable to determine whether esag1 is an essential gene, or what function it fulfils, or whether any competent Esag1 protein is expressed in the mutant trypanosomes.