The nature of spontaneous mutations, including their rate, distribution across the genome, and fitness consequences, is of central importance to biology. However, the low rate of mutation has made it difficult to study spontaneous mutagenesis, and few studies have directly addressed these questions. Here, we present a direct estimate of the mutation rate and a description of the properties of new spontaneous mutations in the unicellular green alga Chlamydomonas reinhardtii. We conducted a mutation accumulation experiment for similar to 350 generations followed by whole-genome resequencing of two replicate lines. Our analysis identified a total of 14 mutations, including 5 short indels and 9 single base mutations, and no evidence of larger structural mutations. From this, we estimate a total mutation rate of 3.23 x 10(-10)/site/generation (95% C.I. 1.82 x 10(-10) to 5.23 x 10(-10)) and a single base mutation rate of 2.08 x 10(-10)/site/generation (95% C.I., 1.09 x 10(-10) to 3.74 x 10(-10)). We observed no mutations from A/T -> G/C, suggesting a strong mutational bias toward A/T, although paradoxically, the GC content of the C. reinhardtii genome is very high. Our estimate is only the second direct estimate of the mutation rate from plants and among the lowest spontaneous base-substitution rates known in eukaryotes.