Two genes, belonging to a new expression site-associated gene family of six to eight members in Trypanosoma equiperdum and Trypanosoma brucei, have been cloned from a T. equiperdum variant. One of them, called ESAG-9c, is contained in the 1.78-C expression site and is found just upstream of the 5' barren region. The other one, called ESAG-9u, is unique in the family, is not telomere linked, and apparently is not expression site related. A 2-kb poly(A)+ mRNA is detected with probes for this ESAG-9 family in all T. equiperdum variants examined. By using polymerase chain reaction and restriction fragment length polymorphism techniques, it has been possible to distinguish between ESAG-9c and ESAG-9u and to show that ESAG-9c is transcribed in an expression site-specific manner. However, ESAG-9u (or another gene in the family having identical characteristics) is transcribed in all variants, regardless of the expression site used by these variants. Thus, this ESAG-9 family contains at least one gene that is under expression site control but might have other genes that are not. The function of these ESAG-9 genes is unknown. Transcripts homologous to ESAG-9 were detected in T. brucei bloodstream forms but not in procyclics.