During in vitro growth Leishmania chagasi promastigotes differentially express 3 classes of RNAs encoding the major surface protease (MSP) gp63 that can be distinguished by their unique 3' untranslated regions. Here we show that the three classes (logarithmic-specific, stationary-specific and constitutively expressed) are encoded by a family of at least 4 tandem stationary genes (mspS2, mspS1, mspS3 and mspS5) followed by twelve or more logarithmic genes (mspL genes), one constitutive gene (mspC) and a final stationary gene (mspS4). Some of the stationary genes can be distinguished from each other by groups of nucleotide differences within the coding regions that result in localized amino acid differences. Northern blots confirm that RNAs from the individual stationary genes are present in stationary, but not logarithmic, phase promastigotes. Western blots using sera directed against synthetic peptides indicate that correspondingly heterogeneous gp63 proteins are expressed in L. chagasi promastigotes. A 200-bp region upstream of all three gp63 gene classes is conserved except for a variable number of 6-bp repeats. Downstream of the gp63 coding regions are highly conserved, class-specific sequences that include the 3' untranslated regions and extend past the polyadenylation site for 65 bp (mspL), 345 bp (mspC) or 2.8 kb (mspS). These sequence features flanking the msp coding regions are likely important in the growth phase-specific expression of the three gp63 RNA classes.