A maximum-likelihood approach to multi-reference image refinement is presented. In contrast to conventional cross-correlation refinement, the new approach includes a formal description of the noise, implying that it is especially suited to cases with low signal-to-noise ratios. Application of this approach to a cryo-electron microscopy dataset revealed two major classes for projections of simian virus 40 large T-antigen in complex with an asymmetric DNA-probe, containing the origin of simian virus 40 replication. Strongly bent projections of dodecamers showed density that may be attributed to the complexed double-stranded DNA, while almost straight projections revealed a twist in the relative orientation of the hexameric subunits. This new level of detail for large T-antigen projections was not detected using conventional techniques. For a negative stain dataset, maximum-likelihood refinement yielded results that were practically identical to those obtained using conventional multi-reference refinement. Results obtained using simulated data suggest that the efficiency of the maximum-likelihood approach may be further enhanced by explicitly incorporating the microscope contrast transfer function in the image formation model.