Abstract Explicit derivations are presented for the expressions necessary to analyze the ESR spectra of low-spin heme iron centers. Emphasis is placed on those centers which satisfy commonly assumed conditions; at the same time, tests for the validity of those assumptions are developed. The means of selecting the correct coordinate system in which to state the results from ESR measurements on powder samples are examined. Furthermore, attention is called to the signs of the principal g values for each center and the limited degree to which these signs are arbitrary. All of the expressions developed are written in a manner intended to facilitate their use. They are illustrated by applying them to data from several categories of hemoglobin derivatives and from ferricytochrome c.