Because spontaneous mutation is the source of all genetic diversity, measuring mutation rates can reveal how natural selection drives patterns of variation within and between species. We sequenced eight genomes produced by a mutation-accumulation experiment in Drosophila melanogaster. Our analysis reveals that point mutation and small indel rates vary significantly between the two different genetic backgrounds examined. We also find evidence that 2% of mutational events affect multiple closely spaced nucleotides. Unlike previous similar experiments, we were able to estimate genome-wide rates of large deletions and tandem duplications. These results suggest that, at least in inbred lines like those examined here, mutational pressures may result in net growth rather than contraction of the Drosophila genome. By comparing our mutation rate estimates to polymorphism data, we are able to estimate the fraction of new mutations that are eliminated by purifying selection. These results suggest that 99% of duplications and deletions are deleterious-making them 10 times more likely to be removed by selection than nonsynonymous mutations. Our results illuminate not only the rates of new small-and large-scale mutations, but also the selective forces that they encounter once they arise.B ECAUSE all genetic variation on which natural selection acts originates via spontaneous mutation, the rate at which various types of mutations appear in natural populations has important consequences for the manner in which organisms evolve. Once we determine the rate at which adaptive and deleterious alleles arise, we can understand how natural selection shapes the spectrum of variation within and between species (Lynch et al. 2008). Mutationaccumulation (MA) experiments are the most widely used method for directly measuring spontaneous mutation rates (Halligan and Keightley 2009). In these experiments genetically identical individuals are subdivided into initially homogeneous sublines, and these sublines are maintained over many generations. At each generation a minimum number of individuals are randomly selected to reproduce and propagate the MA subline, thereby limiting the ability of natural selection to purge new deleterious mutations or to favor new adaptive alleles. Because the MA sublines are initially identical (barring any contamination or residual heterozygosity), any genetic differences among lines must arise via mutations occurring during the course of the experiment.Mutation-accumulation experiments were initially used to assess the phenotypic impacts of mutation (e.g., Mukai et al. 1972). More recently, mutation-accumulation has been used to assess the rate of spontaneous mutation on the genome at specific loci (Mukai and Cockerham 1977), selected genomic regions (Haag-Liautard et al. 2007), or, with the advent of next-generation sequencing technology, genome-wide. Mutation accumulation is now used to estimate genome-wide estimates of per-site mutation rates in a variety of eukaryotes, including Saccharomyces cerevis...