Activation domains (ADs) appear to work by making specific protein-protein contacts with the transcriptional machinery. However, ADs show no apparent sequence conservation, they can be functionally replaced by a number of random peptides and unrelated proteins, and their function does not depend on sustaining a complex tertiary structure. To gain a broader perspective on the nature of interactions between acidic ADs and several of their proposed targets, the in vivo strengths of viral, human, yeast, and artificial activation domains were determined under physiological conditions, and mutant ADs with increased in vivo potencies were selected. The affinities between ADs and proposed targets were determined in vitro and all interactions were found to be of low-level affinity with dissociation constants above 10(-7)M. However, in vivo potencies of all ADs correlated nearly perfectly with their affinities for transcriptional proteins. Surprisingly, the weak interactions of the different ADs with at least two non-transcriptional proteins show the same rank order of binding and AD mutants selected for increased in vivo strength also have increased affinities to non-transcriptional proteins. Based on these results, isolated acidic ADs can bind with relatively low-level specificity and affinity to many different proteins and the strength of these semi-specific interactions determine the strength of an AD. I suggest that ADs expose flexible hydrophobic elements in an aqueous environment to contact hydrophobic patches over short distances, shifting specificity of activators largely to the DNA colocalization of arrays of ADs and targets.