Pleckstrin homology (PH) domains are small protein modules known for their ability to bind phosphoinositides and to drive membrane recruitment of their host proteins. We investigated phosphoinositide binding (in vitro and in vivo) and subcellular localization, and we modeled the electrostatic properties for all 33 PH domains encoded in the S. cerevisiae genome. Only one PH domain (from Num1p) binds phosphoinositides with high affinity and specificity. Six bind phosphoinositides with moderate affinity and little specificity and are membrane targeted in a phosphoinositide-dependent manner. Although all of the remaining 26 yeast PH domains bind phosphoinositides very weakly or not at all, three were nonetheless efficiently membrane targeted. Our proteome-wide analysis argues that membrane targeting is important for only approximately 30% of yeast PH domains and is defined by binding to both phosphoinositides and other targets. These findings have significant implications for understanding the function of proteins that contain this common domain.