Metaphase (from Ancient Greek μετα- (meta-) beyond, above, transcending and from Ancient Greek φάσις (phásis) 'appearance') is a stage of mitosis in the eukaryotic cell cycle in which chromosomes are at their second-most condensed and coiled stage (they are at their most condensed in anaphase). These chromosomes, carrying genetic information, align in the equator of the cell before being separated into each of the two daughter cells. Metaphase accounts for approximately 4% of the cell cycle's duration. Preceded by events in prometaphase and followed by anaphase, microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase.

In metaphase, the centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate), an imaginary line that is equidistant from the two centrosome poles. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochore microtubules, analogous to a tug-of-war between two people of equal strength, ending with the destruction of B cyclin. In certain types of cells, chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly, only roughly lining up along the middleline. Early events of metaphase can coincide with the later events of prometaphase, as chromosomes with connected kinetochores will start the events of metaphase individually before other chromosomes with unconnected kinetochores that are still lingering in the events of prometaphase.

One of the cell cycle checkpoints occurs during prometaphase and metaphase. Only after all chromosomes have become aligned at the metaphase plate, when every kinetochore is properly attached to a bundle of microtubules, does the cell enter anaphase. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase, even if most of kinetochores have been attached and most of the chromosomes have been aligned. Such a signal creates the mitotic spindle checkpoint. This would be accomplished by regulation of the anaphase-promoting complex, securin, and separase.