The L-type calcium current (ICaL) is the first step in cardiac excitation–contraction-coupling and plays an important role in regulating contractility, but also in electrical and mechanical remodeling. Primary culture of cardiomyocytes, a widely used tool in cardiac ion channel research, is associated with substantial morphological, functional and electrical changes some of which may be prevented by electrical pacing. We therefore investigated ICaLdirectly after cell isolation and after 24 h of primary culture with and without regular pacing at 1 and 3 Hz in rat left ventricular myocytes. Moreover, we analyzed total mRNA expression of the pore forming subunit of the L-type Ca2+channel (cacna1c) as well as the expression of splice variants of its exon 1 that contribute to specificity of ICaLin different tissue such as cardiac myocytes or smooth muscle. 24 h incubation without pacing decreased ICaLdensity by ~ 10% only. Consistent with this decrease we observed a decrease in the expression of totalcacna1cand of exon 1a, the dominant variant of cardiomyocytes, while expression of exon 1b and 1c increased. Pacing for 24 h at 1 and 3 Hz led to a substantial decrease in ICaLdensity by 30%, mildly slowed ICaLinactivation and shifted steady-state inactivation to more negative potentials. Totalcacna1cmRNA expression was substantially decreased by pacing, as was the expression of exon 1b and 1c. Taken together, electrical silence introduces fewer alterations in ICaLdensity andcacna1cmRNA expression than pacing for 24 h and should therefore be the preferred approach for primary culture of cardiomyocytes.Graphical Abstract