The neural hormone melatonin (N-acetyl-5-methoxytryptamine) is an indole derivative with a flexible peptide-like side chain that presents five distinct hydrogen-bonding sites for water: the carbonyl oxygen, the amide NH, the indole NH, the methoxy oxygen, and the indole pi cloud. Using a combination of two-color resonant two-photon ionization (2C-R2PI), UV-UV hole-burning spectroscopy, and resonant ion-dip infrared spectroscopy (RIDIRS), the conformational preferences of melatonin upon sequential solvation with water have been examined. Density functional theory calculations are used to identify structural minima and to aid in the infrared spectral assignments. This work builds on previous results on the melatonin monomer (Florio et al. J. Am. Chem. Soc. 2002, 124, 10236), which identified five monomer conformations: three dominant trans-amide conformers and two minor cis-amide conformers. Four distinct melatonin-(water)(1) complexes and two melatonin-(water)(2) clusters are observed. All of these feature water binding to the carbonyl group as the primary point of attachment. The dominant two MEL-(water)(1) complexes retain the structures of the lowest energy monomer conformations, MEL(A, B). The other two structures (MEL-(water)(1) Y and Z) have water bound at the carbonyl group, but also perturb the indole NH stretch fundamental, suggesting that the water molecule interacts with both sites simultaneously. The infrared spectra of the two MEL-(water)(2) clusters point to a bridge structure joining the amide carbonyl and indole NH groups. The changes that occur in the potential energy landscape of melatonin in the presence of water will be discussed.