PINK1 kinase activates Parkin, an E3 ubiquitin ligase, to induce selective autophagy of damaged mitochondria. However, the mechanism by which PINK1 activates and recruits Parkin to mitochondria has been unclear. Although PINK1 phosphorylates Parkin, other PINK1 substrates may also be involved. Using mass spectrometry, the authors discovered that endogenous PINK1 phosphorylates ubiquitin at serine 65, the site phosphorylated by PINK1 in Parkin's ubiquitin-like domain. Recombinant PINK1 directly phosphorylated ubiquitin, and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin, while the ubiquitin mutant S65A, which cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.PINK1 kinase activates Parkin, an E3 ubiquitin ligase, to induce selective autophagy of damaged mitochondria. However, the mechanism by which PINK1 activates and recruits Parkin to mitochondria has been unclear. Although PINK1 phosphorylates Parkin, other PINK1 substrates may also be involved. Using mass spectrometry, the authors discovered that endogenous PINK1 phosphorylates ubiquitin at serine 65, the site phosphorylated by PINK1 in Parkin's ubiquitin-like domain. Recombinant PINK1 directly phosphorylated ubiquitin, and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin, while the ubiquitin mutant S65A, which cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.