To maintain high average fitness, populations must effect selection against the deleterious mutations that continuously arise de novo. Theoretical models of mutation-selection balance predict that the maximum tolerable mutation rate is much lower for organisms growing in colonies than for those in well-mixed liquid media due to drift imposed by competition for position along the growing colony front. Simplifying assumptions made in these models, including the irreversibility and fixed fitness cost of mutations, do not strictly hold in extant species. To explore the applicability of these models in natural contexts, we have constructed a yeast strain which undergoes recombinase-mediated irreversible gene excision at a single locus with tunable fitness cost, but also possesses the random genomic mutation profile characteristic of yeast. We find that several theoretical predictions hold for our strain, including the dependence of maximum tolerable mutation rate on growth condition and selective coefficient. These results constitute the first direct biological test of mutation-selection balance theory.