Objective:To compare two suites of culture media (one with HSA and one protein-free (PF) supplemented with methylcellulose) for washing human sperm in IVF.Methods:Semen samples (n=41) underwent parallel density gradient preparation using PF or HSA-supplemented culture medium and subsequent yield, survival, morphology and motility were compared.Results:The PF medium resulted in a significantly higher sperm yield (P<0.0001), but similar sperm morphology (P=0.822) and 24-hour survival (P=0.11). There was, however, a lower percentage of progressively motile sperm (P<0.0001) and a higher proportion of sperm demonstrating non-progressive motility (P<0.0001) in the PF medium when observed on a Makler Chamber, apparently an artefact as a similar sperm motility index was measured using a Sperm Quality Analyser (P=0.83). Attachment of sperm in PF medium to the glass chamber reduced with time and any differences had disappeared after 6 minutes on the counting chamber.Conclusion:These results support the use of PF media supplemented with methylcellulose as an altative to HSA, although a modification to the manufacturer’s protocol of 6-minutes pre-incubation before assessing sperm motility must be used. Further studies should investigate the function of such sperm prepared in PF medium.