The central region of the matrix protein p17 of HIV-1 is known to be essential during virus assembly. We substituted alanines for amino acid triplets in this region of p17 (amino acid residues 47 to 55: NPG LLE TSE). Introduction of the respective mutations into the gag-coding sequence of HI-proviruses and subsequent transfection into Cos-7 cells led to particle production and release. Exchange of LLE resulted in the production of non-infectious particles. These residues may be important for correct folding and assembly of the processed matrix protein and the production of infectious HIV. In vitro studies of wild-type and mutated matrix proteins using spectroscopic methods (NMR, fluorescence, CD) yielded detailed data about structure and stability. Two-dimensional NMR spectroscopy showed that wild-type and mutant proteins (p17-NPG and p17-TSE) are well folded. Besides structural changes at the mutated site, chemical shift changes indicate small but significant long range structural rearrangements. The stability against chemically and thermally induced unfolding of the mutants p17-NPG and p17-TSE was slightly decreased, while that of p17-LLE was drastically diminished. The alterations have only a local effect on protein folding for the mutants p17-NPG and p17-TSE, and the globular tertiary structure remains nearly unchanged. For p17-LLE, however, the substitutions seem to trigger significant changes in structural elements.