The isoforms of gamma-enolase were characterized in serum from patients with small-cell lung cancer (SCLC) and in extracts from SCLC cell lines and malignant melanoma tumor tissue. Large variations in the expression of the 3 gamma-isoforms of enolase were observed. These forms probably represent the homodimeric gamma gamma-enolase, the heterodimeric alpha gamma-enolase and the monomeric forms of gamma-enolase. Only the dimeric forms are enzymatically active. The predominant gamma-enolase in the cell lines is the heterodimeric alpha gamma-enolase. The SCLC cell lines can be divided into two groups: one with negligible gamma gamma-enolase expression and considerable amounts of the nonneuronal alpha alpha-enolase and a second group with a large fraction of gamma gamma-enolase concomitant with a low expression of alpha-enolase. Similar patterns are observed in tissue extracts from malignant melanoma. When changing buffer conditions by increasing the ionic strength and decreasing the Mg2+ concentration, interconversions between the isozymes occur. In contrast to the predominant alpha gamma-enolase in extracts from cell lines, the multiple forms of gamma-enolase in serum might be caused by a subunit exchange facilitated by the low Mg2+ concentration in plasma. However, there seems to be a stable equilibrium between the isoforms in undiluted patient serum. The induction of subunit exchange by perturbation in ionic strength and/or Mg2+ concentration indicates a need for caution when choosing diluents for use in assays for neuron-specific enolase.