Using a highly sensitive pulsed-flow microcalorimeter, we have measured the changes in enthalpy and determined the thermodynamic parameters DELTA-H, DELTA-S-degrees, DELTA-G-degrees, and DELTA-C(p) for Cro protein-DNA association reactions. The reactions studied include sequence-nonspecific DNA association and sequence-specific DNA associations involving single- and multiple-base alterations and/or single-amino acid alteration mutants. (i) The association of Cro protein with nonspecific DNA at 15-degrees-C is characterized by DELTA-H = +4.4 kcal.mol-1 (1 cal = 4.18J), DELTA-S-degrees = 49 cal.mol-1.K-1, DELTA-G-degrees = -9.7 kcal.mol-1, and DELTA-C(p) congruent-to 0; the association this specific high-affinity operator OR3 DNA is characterized by DELTA-H = +0.8 kcal.mol-1, DELTA-S-degrees 59 cal.mol-1.K-1, DELTA-G-degrees = -16.1 kcal.mol-1, and DELTA-C(p) = -360 cal.mol-1.K-1, respectively. Both nonspecific and specific Cro-DNA associations are entropy-driven. (ii) Plots of DELTA-H vs. DELTA-C(p) and DELTA-S-degrees vs. DELTA-C(p) for the 20 association reactions studied fall into two correlation groups with linear slopes of +9.4 K and -20.5 K and of -0.03 and -0.14, respectively. These regression lines have common intercepts, at the DELTA-H and DELTA-S-degrees values of nonspecific association (where DELTA-C(p) congruent-to 0). The results suggest that there are, at least, two distinct conformational subclasses in specific Cro-DNA complexes, stabilized by different combinations of enthalpic and entropic contributions. The DELTA-G-degrees and DELTA-C(p) values form an approximately single linear correlation group as a consequence of compensatory contributions from DELTA-H and DELTA-S-degrees to DELTA-G-degrees and to DELTA-C(p). Cro protein-DNA associations share some similar thermodynamic properties with protein folding, but their overall energetics are quite different. Although the nonspecific complex is stabilized predominantly by electrostatic forces, it appears that H bonds, van der Waals contacts, hydrophobic effects, and charge interactions all contribute to the stability (DELTA-G-degrees and DELTA-C(p)) of the specific complex. (iii) The variations in the values of the thermodynamic parameters are in general accord with our knowledge of the structure of the Cro-DNA complex.