We aim to study the effects of activity distribution for multiplexing multi-pinhole (MPH) SPECT. Three digital phantoms, including a hot rod, a cold rod and a cold sphere phantom, were used. Different degrees of multiplexing were obtained by (i) adjusting the MPH pattern for the same 4-pinhole collimator (scheme 1) and (ii) increasing the number of pinholes (scheme 2). Noise-free and noisy projections were generated using a 3D analytical MPH projector based on the same acquisition time. Projections were reconstructed using OS-EM without resolution recovery. Normalized mean-square-error (NMSE), noise, image profiles and signal-to-background ratios (SBR) were assessed. For the hot rod phantom, the NMSE-noise trade-offs slightly improves for multiplexing designs in scheme 2. Substantial artifacts were observed and the NMSE-noise trade-offs slightly worsened for multiplexing designs for the cold phantoms. Resolutions slightly degraded for higher degrees of multiplexing (∼39-65%) for the cold rod phantom. For the cold sphere phantom, image profiles showed non-multiplexing designs better emulated the phantom, while ∼20% multiplexing performs similarly as compared to non-multiplexing in SBR. Our results indicate that multiplexing can help for sparse objects but leads to a significant image degradation in non-sparse distributions. Since many tracers are not highly specific, and the gain of detection efficiency by allowing multiplexing is fairly offset by image degradations, multiplexing needs to be kept to a minimum for optimum MPH collimator designs. © 2011 Institute of Physics and Engineering in Medicine.