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AIMS: To estimate the prevalence of and risk factors associated with concurrent injection of multiple substances (co-injection) among a community-recruited cohort of people who inject drugs. DESIGN: Cross-sectional study. SETTING: Melbourne, Australia. PARTICIPANTS: A sample of 720 actively injecting participants from the Melbourne Injecting Drug User Cohort Study (33% female) was extracted. MEASUREMENTS: We constructed two statistical models: a logistic regression model analysing correlates of co-injection of any substance combination in the past month and a multinomial logistic regression model analysing correlates of three mutually exclusive groups: heroin-diphenhydramine co-injection only, co-injection of other substances and no co-injection. Risk factors examined included drug use characteristics, demographic characteristics, health service use, hepatitis C status, injection risk behaviours and previous experience of non-fatal overdose. FINDINGS: One-third (n=226, 31%; 95% CI: 28-34%) of participants reported co-injecting substances within the past month, with equal numbers of participants reporting injecting combinations of heroin-diphenhydramine (n=121, 54%; 95% CI: 48-60%) and heroin-methamphetamine (n=121, 54%; 95% CI: 48-60%). In logistic regression analyses, reporting co-injection of any substance combination was associated with male sex (AOR: 1.80, 95% CI: 1.18-2.74, p=0.006) and injecting daily or more frequently (AOR: 2.04, 95% CI: 1.31-3.18, p=0.002). In multinomial logistic regression analyses, participants reporting heroin-diphenhydramine co-injection only were significantly more likely to report groin injecting (ARRR: 6.16, 95% CI: 2.80-13.56, p<0.001) and overdose (requiring an ambulance) in the past 12 months (ARRR: 2.81, 95% CI: 1.17-6.72, p=0.021) compared with participants reporting no co-injection or co-injection of other substances. CONCLUSIONS: A substantial proportion of people who inject drugs report co-injection of multiple substances, which is associated with a range of socio-demographic, drug use and health service use risk factors.