Dopamine is a neuromodulator that regulates behavior: it is critical for movement and its function has been linked to how the brain processes relevant information that carries the value of a reward. While together these functions are crucial for the expression of goal-directed behavior, it is not clear how are they encoded at the cellular level. One possibility is that dopamine neurons are functionally heterogeneous and receive selective information from brain areas associated with movement and motivational functions. In this paper we asked what drives the activity of dopamine neurons. Specifically, we looked at the modulation by acetylcholine, a neuromodulator typically associated with attention and learning. Using novel technologies for selectively controlling the activity of cholinergic neurons, we found that acetylcholine provides an activatory signal to dopamine neurons mediated by direct synaptic communication. Notably, the nature of this signal and its effects on dopamine neurons is distinct depending on whether it originates from motor systems or from limbic (motivational) systems. The differential effects on dopamine neurons were observed to elicit distinct behavioral responses and depend on the activation of nicotinic receptors. Our results reveal that motor and motivational signals are conveyed to dopamine neurons by segregated acetylcholine signals and likely act together to produce an integrated behavioral output. Our data thus contribute to identify how the activity of dopamine neurons is regulated, which is key to understand their involvement in a variety of conditions such as Parkinson's disease and addiction.