VP1, the major coat protein of polyomavirus, assembles intracellularly to virus-like particles if expressed in eukaryotes. Here, the nonconventional yeast Kluyveromyces lactis was used for production of virus-like particles of murine polyomavirus. The heterologous gene of VP1 was integrated in the LAC4 locus of the GAL/LAC genes. Consequently the expression of VP1 is regulated by the interplay of the activator KlGal4p and inhibitor KlGal80p. This cloning strategy couples the production of VP1 to that of the enzyme β -galactosidase, allowing a fast alternative for monitoring the course of recombinant protein production by measuring the β -galactosidase activity. A Klgal80 knockout strain was generated for a constitutive expression of VP1 and a continuous VLP production. High-cell-density fermentation showed that (1) Kluyveromyces lactis is generally suitable for VLP production and (2) the Klgal80 knockout strain produces higher amounts of recombinant VP1. Furthermore, VLPs could be purified chromatographically to 87% (w/w) of total protein, and showed a homogeneous species of 45-nm particles and a high resistance against proteolysis compared to conventional in vitro assembled VLPs. This demonstrates the superior stability of virus-like particles produced in yeast.