To improve toughness and fatigue strength, a mutiphase (ferrite (F)-bainite (B)-martensite (M)) microstructure was developed in a V-bearing medium carbon microalloyed (MA) steel through a two-step cooling process that was followed by an annealing (two-step cooling and annealing (TSCA)) treatment. In the present paper, the high cycle fatigue (HCF) response determined in terms of the endurance limit, long crack fatigue threshold (Δ K th), crack closure and fatigue crack growth rate (FCGR) in a material that has a multiphase microstructure is presented and compared with those of the same material with a ferrite-pearlite (F-P) and a tempered martensite (T-M) microstructure obtained by air-cooling (AC) and quenching and tempering (Q&T), respectively. Long crack fatigue threshold (Δ K th) and crack closure were evaluated using a dynamic compliance (DYNACOMP) measurement technique. The fatigue limit of the F-B-M and the T-M microstructures (∼400 MPa) was greater than that of the F-P microstructure (∼340 MPa). At load ratios less than 0.5, the threshold for long crack growth was lower for the F-B-M microstructure compared with that of the F-P microstructure. This is attributed to the reduced roughness-induced crack closure (RICC) contribution to the threshold in the former multiphase microstructure. A quantitative analysis of the near-threshold fracture surfaces validated the above conclusion. Fatigue crack growth rate in the Paris regime was found to be independent of the microstructure but dependent on the load ratio. © 2003 Elsevier B.V. All rights reserved.