The difference between natural and cultured pearls focuses on whether the pearl was created spontaneously by nature — without human intervention — or with human aid. Pearls are formed inside the shell of certain mollusks: as a defense mechanism to a potentially threatening irritant such as a parasite inside its shell, the mollusk creates a pearl to seal off the irritation.

Natural pearls are nearly 100% calcium carbonate and conchiolin. It is thought that natural pearls form under a set of accidental conditions when a microscopic intruder or parasite enters a bivalve mollusk, and settles inside the shell. The mollusk, being irritated by the intruder, secretes the calcium carbonate and conchiolin to cover the irritant. This secretion process is repeated many times, thus producing a pearl. Natural pearls come in many shapes, with perfectly round ones being comparatively rare.

Cultured pearls (nucleated and non-nucleated or tissue nucleated cultured pearls) and imitation pearls can be distinguished from natural pearls by X-ray examination. Nucleated cultured pearls are often 'pre-formed' as they tend to follow the shape of the implanted shell bead nucleus. Once the pre-formed beads are inserted into the oyster, it secretes a few layers of nacre around the outside surface of the implant before it is removed after six months or more.

When a nucleated cultured pearl is X-rayed, it reveals a different structure to that of a natural pearl. A cultured pearl shows a solid center with no concentric growth rings, whereas a natural pearl shows a series of concentric growth rings.