Water-soluble hormones cannot cross the plasma membrane, so they rely on protein receptors that span the membrane to trigger intracellular signaling pathways. These pathways then activate second messengers inside the cell, including cAMP or calcium ions.

Many hormones bind to transmembrane G protein-coupled receptors that connect to regulatory G proteins. These G proteins can then activate enzymes such as adenylyl cyclase or phospholipase C. Adenylyl cyclase converts ATP to cAMP, activating protein kinases. Kinases are enzymes that add phosphate groups to other proteins, initiating a phosphorylation cascade. Epinephrine, glucagon, parathyroid hormone, and luteinizing hormone all act through cAMP. For instance, epinephrine activates the β-adrenergic receptor, initiating the GPCR signaling cascade to regulate the "fight or flight" response.

Phospholipase C, conversely, breaks down the membrane phospholipid PIP2 into IP3 and DAG. IP3 then moves to the endoplasmic membrane and attaches to an IP3-gated calcium channel, which causes a release of calcium ions into the cytoplasm. The change in calcium concentration can induce physiological effects such as muscle contraction. Oxytocin and hypothalamic regulatory hormones amplify signals through PIP2 and calcium ions.