Initial playback of recorded birdsong triggers a number of responses in zebra finches, including overt listening behavior and ERK pathway-dependent activation of zenk gene transcription in the auditory lobule of the forebrain. Repetition of one song stimulus leads to persistent habituation of these responses, as measured by subsequent presentations 1 day later. In this study, we examined the causal relationships between behavioral and molecular (ERK/zenk) habituation. In a within-subject comparison, we found a strong correlation with the level of prior training for both responses (duration of behavioral listening and magnitude of zenk expression), but little correlation between these responses for birds within the same treatment group. We then tested the hypothesis that ERK/zenk activation during training is necessary for the development of habituation measured 1 day later. Cannula-directed infusion of a pharmacological inhibitor of ERK activation (U0126) immediately before training blocked the development of habituation of the zenk gene response. However, measurement of the effect on behavioral habituation was confounded because birds that were infused with a non-active drug analogue (U0124) showed a decreased response 1 day later, even to novel songs. We conclude that the behavioral response to song stimulation is strongly influenced by factors other than song familiarity, whereas the zenk response in the forebrain may be a more accurate indicator of actual experience hearing a particular song.