Abstract Spontaneous mutations are the source of new genetic variation and are thus central to the evolutionary process. In molecular evolution and quantitative genetics, the nature of genetic variation depends critically on the distribution of fitness effects (DFE) of mutations. Spontaneous mutation accumulation (MA) experiments have been the principal approach for investigating the overall rate of occurrence and cumulative effect of mutations, but have not allowed the effects of individual mutations to be studied directly. Here, we crossed MA lines of the green alga Chlamydomonas reinhardtii with its unmutated ancestral strain to create haploid recombinant lines, each carrying an average of 50% of the accumulated mutations in a variety of combinations. With the aid of the genome sequences of the MA lines, we inferred the genotypes of the mutations, assayed their growth rate as a measure of fitness, and inferred the DFE using a novel Bayesian mixture model that allows the effects of individual mutations to be estimated. We infer that the DFE is highly leptokurtic (L-shaped), and that a high proportion of mutations increase fitness in the laboratory environment. The inferred distribution of effects for deleterious mutations is consistent with a strong role for nearly neutral evolution. Specifically, such a distribution predicts that nucleotide variation and genetic variation for quantitative traits will be insensitive to change in the effective population size.