As part of the Trypanosoma cruzi Genome Initiative, we have mapped a large portion of the chromosomal bands XVI (2.3 Mb) and XVII (2.6 Mb) containing the highly repetitive and immunodominant antigenic gene families h49 and jl8. Restriction mapping of the isolated chromosomal bands and hybridization with chromosome specific gene probes showed that genes h49 and jl8 are located in a pair of size-polymorphic homologous chromosomes. To construct the integrated map of the chromosomes harboring the h49 and jl8 loci, we used YAC, cosmid, and lambda phage overlapping clones, and long range restriction analysis using a variety of probes (i.e., known gene sequences, ESTs, polymorphic repetitive sequences, anonymous sequences, STSs generated from the YAC ends). The total length covered by the YAC contig was approximately 670 kb, and its map agreed and was complementary to the one obtained by long-range restriction fragment analysis. Average genetic marker spacing in a 105 kb region around h49 and jl8 genes was estimated to be 6.2 kb/marker. We have detected some polymorphism in the H49/JL8 antigens-encoding chromosomes, affecting also the coding regions. The physical map of this region, together with the isolation of specific chromosome markers, will contribute in the global effort to sequence the nuclear genome of this parasite.