Objective To conduct a cost minimisation analysis of three methods of gestational diabetes mellitus (GDM) screening and diagnosis.Design Prospective randomised controlled trial.Setting University teaching hospital.Population Pregnant women (n = 1594) presenting for GDM screening.Methods Women presenting for GDM screening, who consented to participate, were randomised to GR1 [1-hour, 50-g glucose screen (GS) ± 3-hour, 100-g oral glucose tolerance test (OGTT)], GR2 (50-g GS ± 2-hour, 75-g OGTT) or GR3 (2-hour, 75-g OGTT). Demographics, health and time/travel cost information were assessed for each glucose testing visit.Main outcome measures Costs (direct and indirect) and prevalence of GDM diagnosis.Results The direct sampling costs of the glucose tests per woman were as follows: GS, CAN$12.57; 75-g OGTT, $36.10; 100-g OGTT, CAN$48.13. Among women in the two-step method groups diagnosed with GDM, 39% of the GR1 and 61% of the GR2 groups were diagnosed at the first step by GS ‡ 10.3 mmol/l, according to the Canadian Diabetes Association recommendations, contributing to a lower total cost in these groups. The total costs per woman screened were as follows: GR1, CAN$91.61; GR2, CAN$89.03; GR3, CAN$108.38. The GDM prevalence was similar (3.7%, 3.7% and 3.6%, respectively). The higher costs of GR3 were related to more blood draws and the time required for all women to undergo the 2-hour OGTT.Conclusions Careful consideration should be given to an internationally recommended method of universal screening for GDM which minimises the burden and cost for individual women and the healthcare system, yet provides diagnostic efficacy. The two-step method (GS ± OGTT) accomplished this better than the one-step method (75-g OGTT).Keywords Cost analysis, diagnosis, direct/indirect costs, gestational diabetes mellitus, oral glucose tolerance test, screening.Please cite this paper as: Meltzer S, Snyder J, Penrod J, Nudi M, Morin L. Gestational diabetes mellitus screening and diagnosis: a prospective randomised controlled trial comparing costs of one-step and two-step methods. BJOG 2010;117:407-415.