The objective of this study was to determine the molting process of Dirofilaria immitis third-stage larvae (L-3) to fourth-stage larvae (L-4), as it occurred in vitro. After 48 hr in vitro, the L-4 epicuticle was completely formed, and by 72 hr there was a clear separation between the L-3 and L-4 cuticles. The thickness of the newly formed L-4 cuticle was significantly less than that which has been described for larvae recovered from dogs after a similar incubation time period. If culture conditions were lacking in bovine albumin or proper temperature, larvae successfully developed the L-4 epicuticle but did not complete ecdysis. The molting process of D. immitis L-3 was thus shown to be multistepped with different factors required to induce the various developmental phases.